Agricultural ecosystems play an important role in the storage and release of C within the terrestrial carbon cycle. A study was conducted in north Alabama in 2003-2006 to measure CO2 efflux and carbon storage in long-term tilled and non-tilled cotton plots receiving poultry litter and ammonium nitrate as N sources. Treatments were established in 1996 and consisted of conventional-tillage (CT), mulch-tillage (MT), and no-tillage (NT) systems with winter rye [Secale cereale (L.)] cover cropping and ammonium nitrate (AN) and poultry litter (PL) as nitrogen sources. In 2003, 2004 and 2006 cotton was planted and in 2005 corn was planted as a rotation crop using a no till planter in all plots and did not receive any fertilizer. All tillages with PL application recorded higher CO2 emission from soil compared to AN application. In 2003 and 2006, CT (4.39 and 3.40 ėmol m-2 s-1, respectively) and MT (4.17 and 3.39 ėmol m-2 s-1, respectively) with PL @ 100 kg ha-1 N (100 PLN) recorded significantly higher CO2 efflux compared to NT with 100 PLN (2.84 and 2.47 ėmol m-2 s-1, respectively). On average, CT and MT with 100 PLN emitted 37 and 25%, respectively higher CO2 compared to NT with 100 PLN. In all years cotton–rye cropping system (CR) recorded higher CO2 efflux compared to cotton-fallow (CF). CT and MT with CR released higher CO2 from soil compared to NT with CR in 2003 and 2006. Total carbon in soil (0-15cm) did not differ significantly with tillage. Our study suggests that NT conservation tillage systems along with application of poultry litter @ 100 kg N ha-1 and winter rye cover cropping emits lower CO2 in to the atmosphere compared to conventional and mulch till. Further it helps in safe disposal of poultry litter which is a major problem in southeastern US.