Milk synthesis is a complex biological process that occurs in mammary alveoli to meet nutritional requirements of the offspring. Lactose made of glucose and galactose is the major carbohydrate in milk contributing to its energy value. Lactose is also the principal osmole drawing water into milk, and therefore, lactose production is the main determinant of milk volume having desired specific gravity. Enhancing lactose synthesis in dairy cows would be advantageous to dairy industry as the price of nearly 40% of cow milk produced in the US is determined based on milk volume. On the other hand, circumstances, such as COVID-19 would create a situation, where farmers are asked to cut back on production to mitigate the dumping of milk. Moreover, production of milk oligosaccharide having various health benefits are shown to be closely linked with lactose synthesis and lactose concentration in milk. Therefore, understanding the factors governing lactose synthesis is key to develop strategies for manipulating milk yield and its value. Essential amino acids (EAA) are hypothesized to be one of those factors as supplementation not only increases milk protein yield but also the lactose yield in dairy cows. Among EAA, branched-chain amino acids (BCAA) including leucine (Leu), isoleucine (Ile), and valine (Val) have been garnered special interest because of their abilities to elicit anabolic signals in mammary and other cells. In muscle and splanchnic tissues, Leu and Ile supply have been shown to enhance the abundance of facilitated glucose transporter 1 (GLUT1), the most prevalent glucose transporter in lactating mammary glands of dairy cows. Glucose uptake into milk secretory cells is considered as a rate limiting step of lactose synthesis. Once taken into the cells, a portion of glucose is converted to galactose, which is then transported into Golgi apparatus, where lactose synthesis takes place. The abundance of some enzymes involved in derivation of galactose and lactose synthesis have also been identified to be rate limiting for lactose synthesis. Therefore, the primary objective of this study was to determine the effects of BCAA on lactose synthesis pertaining to glucose uptake, derivation of galactose from glucose, and fractional rate of lactose synthesis (FSR) in cow mammary cells and mammary tissues. Acknowledging the knowledge gap in scientific literature, we also aimed at exploring the contribution of galactose, not derived from glucose, to lactose synthesis in the present study. A series of in vitro experiments using primary bovine mammary epithelial cells (BMEC) and mammary tissue explants from lactating cows (MTS) were conducted. The abundance of GLUT1 and the enzymes were determined using Western immunoblotting and proteomic analysis. The FSR and contribution of extracellular galactose to lactose synthesis in MTS were determined using 13C-labeled glucose and galactose, and GC-MS analysis. The abundance of GLUT1 in the cell membrane of BMEC increased significantly (P < 0.001) for extracellular total BCAA deficiency (-BCAA). The degree of glycosylation of GLUT1 positively related to its activity also increased significantly (P = 0.031) for –BCAA. Among individual BCAA, only Leu deficiency increased the abundance of GLUT1 in the membrane and glucose uptake (P < 0.050), whereas Ile or Val had no significant impact on any of those responses in BMEC. The total BCAA deficiency was positively associated with the abundance of hexokinase (75%, P = 0.065) that closely operates with GLUT1. On the other hand, -BCAA was negatively related to the abundance of β-1, 4-galactosyltransferase (P = 0.069) that catalyzes the lactose synthesis reaction in Golgi apparatus. In MTS, -BCAA did not change 13C enrichment in free precursor pools (P = 0.755) but decreased the enrichment in lactose-bound precursor pool (6.55 vs 8.66%, P = 0.022) and FSR (2.59 vs. 3.37 %/h, P = 0.040) suggesting a significant involvement of BCAA, individually or together, in regulating precursor utilization efficiency downstream of lactose synthesis pathway. On the contrary, deprivation of Leu alone did not change any of those parameters (P > 0.510). At a supraphysiological concentration (3.15 mM), extracellular galactose contributed to only 5% of free glucose pool and 8% of lactose-bound precursor pool in MTS (P < 0.050). The contribution of galactose to free glucose became more significant (P = 0.004 vs 0.076), when extracellular Leu concentration was raised from physiological concentration (0.1 mM) to a supraphysiological (2.0 mM) concentration. Extracellular Leu increasing from 0 to 2.0 mM was positively related to the abundance of UDP-galactose-4-epimerase (GALE) catalyzing the conversions of galactose to glucose. Nonetheless, extracellular Leu rising further up to 5.0 mM decreased the abundance of GALE (P = 0.039). Moreover, the contribution of extracellular galactose to lactose synthesis was significantly dependent on glucose supply to MTS as % of free and lactose-bound precursors originating from extracellular galactose became insignificant (P > 0.510), when extracellular glucose concentration decreased from 3.2 to 0.32 mM. Overall, extracellular BCAA availability was negatively and positively related to glucose uptake and lactose synthesis rates, respectively. Despite the significant control over glucose uptake, negligible response of lactose synthesis rates to extracellular Leu supports the notion that the glucose uptake might not be as rate-limiting as the events involved downstream of lactose synthesis pathway. Therefore, future studies focused on Golgi protein-specific analysis would help improve our understanding about the roles of BCAA in regulating lactose synthesis. The results also pointed out that all BCAA rather than simply Leu might be critical in regulating lactose synthesis rates. Therefore, future studies aiming at exploring the effects of all three BCAA, individually and in different combinations could be recommended. In the present study, we demonstrated that mammary tissue slices from lactating cows, stable isotope-labeled glucose, and GC-MS analysis together provide a good model to study lactose synthesis in vitro. However, accounting for the variabilities of tissues within (e.g., front vs hind quarters) and between cows (e.g., parity and stage of lactation) would help further optimize this model.