Deciphering the function of genes that impact fatty acid metabolism is critical to understanding the complex mechanisms of fatty acid and lipid accumulation in plants. Here we report crystal structures, ligand-binding properties, and in vivo functional characterization of a non-catalytic CHI-fold family from plants. Arabidopsis thaliana contains five actively transcribed CHI-fold genes, three of which additionally encode amino-terminal chloroplast-transit sequences. These three CHI-fold proteins localize to plastids, the site of de novo fatty acid biosynthesis in plant cells. Furthermore, their expression profiles correlate with those of core FA biosynthetic enzymes, with maximal expression occurring in seeds and coinciding with increased FA storage in the developing embryo. In vitro, these proteins are Fatty Acid-binding Proteins (FAP). FAP knockout A. thaliana plants show elevated alpha-linolenic acid levels and marked reproductive defects, including aberrant seed formation. Furthermore, polar lipids and fatty acids were quantified in seedlings and seeds of wild type and fap single and double mutants using tandem ionization mass spectrometry (GC-MS/MS) and gas chromatography mass spectrometry (GC-MS). Polar lipid profiling uncovers alterations of the levels of multiple lipid classes, as well as changes in specific molecular species in comparison to levels of these constituents in wild-type controls; in particular MGDG, DGDG, PC, PE, and LPE classes are increased in most fap seedlings, whereas PA, PI, LPG, and LPC are altered in fap seeds. These data led us to postulate that the alteration in polar lipids might occur at a transcriptional level, i.e. that reducing expression of the FAP genes might result in changes in expression of fatty acid and lipid metabolism genes. RNA sequencing analysis of seedlings and developing siliques of wild-type and fap single and double mutants shows that 37 genes encoding proteins involved in the subsequent steps of fatty acid metabolism, including acyltrasferases, desaturass, and elongases exhibit lower levels of expression in all fap mutants. Interestingly, many genes down-regulated in fap seedlings are associated with defense and signaling responses, and the majority of genes down-regulated in fap siliques encode proteins related to seed and embryo maturation (www.arabidospsis.org, www.metnetdb.org; Mentzen and Wurtele, 2008). However, many genes up-regulated in fap mutants encode proteins of unknown function that represent about one third of the Arabidopsis genome.