The increase in atmospheric carbon dioxide (CO2) over the last century has led to an increased interest in soil C dynamics and sequestering C in soil. The focus of this study is to investigate CO2 emissions and soil C changes with various N application rates in a corn-soybean rotation. The study consists of seven sites across Iowa. Each experiment consists of four selected N rates of 0, 90, 180 and 225 kg ha−1 replicated four times in a randomized complete block design in a corn-soybean rotation. Soil samples were collected at 0-5, 5-10, 10-15, 15-30 and 30-60 cm increments for soil organic carbon (SOC), total soil N (TN), particulate organic matter carbon (POMC), and associated mineral fraction C (MFC). Field soil CO2 emissions from the same N rate treatments were measured during the growing season using a Li-Cor 6400 Infrared Gas Analyzer every seven to 10 days at the Boone, Floyd, and Warren sites in 2002, and Tama site in 2003. Soil laboratory incubation, microbial biomass, and N mineralization studies were conducted on the soils from Boone, Floyd, and Tama sites. Results indicated cumulative soil CO2-C emissions varied in response to N fertilization. Higher N rate treatments had lower cumulative CO2-C than the 0 kg ha−1 N rate treatment when N fertilizer was applied to the corn crop. However, N fertilizer rates of 90, 180 and 225 kg ha−1 N applied to corn the previous year showed higher cumulative CO2-C emissions than the 0 kg ha−1 N rate treatment in the soybean season. In a laboratory soil incubation study, N fertilized soils emitted significantly less CO2-C than the non-fertilized soils. Nitrogen fertilization significantly increased inorganic soil N concentration prior to a 56-day laboratory incubation. After the 56-day incubation period, inorganic soil N concentration was significantly lower for the 0 kg ha−1 N rate treatments than the higher N rate treatments. Nitrogen fertilization did not have a significant effect on microbial biomass carbon (MBC). Results indicate N fertilization does not significantly affect soil SOC, POMC, or MFC at the 0-5, 5-10, and 15-30 cm depths. Nitrogen fertilization had variable effects on crop biomass production along with TC and TN potentially returned to the soil via biomass. Overall, after 2 years of N fertilizer application to corn and one year of soybean with no N fertilizer application, no significant changes in soil SOC, POMC, and MFC were observed in the short period of time.