Central to recent scientific debate concerning land conversion to various biofuel crop production systems is whether the new system will be ecologically, economically, and socially sustainable. Impacts on ecosystem carbon (C), energy balance, and greenhouse gas emissions are important considerations if biofuel production is implemented at large scales. Here we established a field experiment and deployed a cluster of eddy-covariance towers to quantify the magnitude and changes of ecosystem carbon assimilation, loss, and balance (i.e, Net Ecosystem Production) in an intact prairie and three types of candidate biofuel crop production systems [Conservation Reserve Program (CRP) grassland, Switchgrass, and mixed-species native prairie] that were converted from agricultural crops or CRP land. The six large fields were converted to soybean in 2009 before establishing the biofuel cropping systems in 2011. Our field observations made between January 2009 (i.e., pre-conversion) through December 2010 showed that conversion of CRP to soybean induced net C emissions (denoted as positive) of 155.7 (±25) - 128.1 (±27) g C m^-2 yr^-1, while at the reference CRP grassland the NEP was -42.9 to – 16.1 g C m^-2 yr^-1 in 2009 and 2010, respectively. For 2010, the C balance and loss via respiration were mostly related to previous land use. Corn cultivation induced a net C accumulation of  310 and 220 g C m^-2 year^-1 , resulting in a C balance of  -42 g C  m^-2  over the 2 years at the sites previously cultivated as CRP, and -340  g C m^-2 at the site under corn-soybean rotation. The CRP grassland sites converted to biofuel crop production have shown a net emission of C over the 2 years, ranging from 303 to 281 g C m^-2.  The conversion of CRP lands has thus incurred a “carbon debt” during the first two years that can take many years to repay. Our results demonstrate the importance of antecedent land use as well as ensuing biofuel crop management in controlling the net carbon balance of the ecosystem.