Poultry litter, an inexpensive source of nutrients and available in abundant amounts in the southeastern USA, may increase N cycling and reduce N losses compared with inorganic N fertilization if applied for crop production. We evaluated the effect of long‐term application of poultry litter and inorganic N fertilizer on crop N uptake, soil N fractions, and N losses in conservation and conventional tillage with or without cover crop at the 0‐ to 20‐cm depth in a Decatur silt loam (clayey, kaolinitic, thermic, Typic Paleudults) in northern Alabama. Treatments were incomplete factorial combinations of three tillage practices [no‐till (NT), mulch till (MT), and conventional till (CT)], two cropping systems [cotton (Gossypium hirsutum L.)–cotton–corn (Zea mays L.) and rye (Secale cereale L.)/cotton–rye/cotton–corn], and two N fertilization sources and rates (0 and 100 kg N ha−1 from NH4NO3 and 100 and 200 kg N ha−1 from poultry litter). Nitrogen fractions were soil total N (STN), particulate organic N (PON), microbial biomass N (MBN), potential N mineralization (PNM), NH4–N, and NO3–N. Crop N uptake varied among treatments and years and total uptake from 1997 to 2005 was greater in rye/cotton–rye/cotton–corn than in cotton–cotton–corn and greater with NH4NO3 than with poultry litter at 100 kg N ha−1 After 10 yr, STN and PON contents at 0 to 20 cm were greater in NT with poultry litter than in other treatments, except in CT with poultry litter, resulting in N storage at 38 kg N ha−1 yr−1 with poultry litter compared with loss at 4 kg N ha−1 yr−1 with NH4NO3. The MBN, PNM, and NO3–N contents were greater with poultry litter than with NH4NO3, regardless of tillage. Calculation of estimated N balance showed that poultry litter conserved N at 191 to 556 kg N ha−1 compared with –75 to 201 kg N ha−1 with NH4NO3. Poultry litter application can increase soil N storage and mineralization and reduce the potential for N loss compared with inorganic N fertilization, thereby reducing the need for N fertilization and environmental N contamination.