Switchgrass (Panicum virgatum L.) grown for biomass feedstock production has the potential to increase soil C sequestration, and soil CO2 flux in grassland is an important component in the global C budget. The objectives of this study were to: (1) determine the effects of N fertilization and harvest frequency on soil CO2 flux, soil microbial biomass carbon (SMBC), and potentially mineralizable carbon (PMC); and (2) evaluate the relationship of soil CO2 flux with soil temperature, soil moisture, SMBC, and PMC. Two N rates (0 and 224kgha−1) were applied as NH4NO3 and cattle (Bos Taurus L.) manure. Switchgrass was harvested every year at anthesis or alternate years at anthesis. The data were collected during growing season (May–October) 2001–2004 on switchgrass-dominated Conservation Reserve Program (CRP) land in east-central South Dakota, USA. Manure application increased soil CO2 flux, SMBC, and PMC during the early portion of the growing season compared with the control, but NH4NO3 application did not affect soil CO2 flux, SMBC, and PMC. However, seasonal variability of soil CO2 flux was not related to SMBC and PMC. Estimated average soil CO2 fluxes during the growing periods were 472, 488, and 706g CO2–Cm−2 for control, NH4NO3–N, and manure-N plots, respectively. Switchgrass land with manure application emitted more CO2, and approximately 45% of the C added with manure was respired to the atmosphere. Switchgrass harvested at anthesis decreased soil CO2 flux during the latter part of the growing season, and flux was lower under every year harvest treatment than under alternate years harvest. Soil temperature was the most significant single variable to explain the variability in soil CO2 flux. Soil water content was not a limiting factor in controlling seasonal CO2 flux.