Long‐term tillage and manure application can alter a soil's ability to sequester nutrients and mineralize C and N. A laboratory incubation study (C and N mineralization) evaluated the long‐term impact of poultry litter (PL) application (>10 yr) and tillage practice (>25 yr). Soil chemical properties (pH, total C, total N, and Mehlich‐1 extractable P, K, Ca, and Mg) were also assessed. Soil was collected (0–5‐, 5–10‐, and 10–20‐cm depths) from continuous soybean [Glycine max (L.) Merr.] and corn (Zea mays L.) systems managed under conventional tillage (CT) or no‐till (NT) with either PL or inorganic fertilizer (IF) applications. The study was located in northeast Alabama on a Hartsells fine sandy loam (a fine‐loamy, siliceous, subactive, thermic Typic Hapludult). Poultry litter and NT increased soil nutrients (N, P, K, Ca, and Mg), primarily at the 0‐ to 5‐cm depth. No‐till concentrated nutrients near the soil surface as opposed to the more even distribution seen under CT. The NT‐PL treatment had higher soil C for corn and soybean (2.25 and 1.83 g kg−1 C, respectively), followed by NT‐IF (1.73 and 1.11 g kg−1 C, respectively). Carbon and N mineralization was higher at the 0‐ to 5‐cm depth for NT and CT compared with lower depths. Long‐term PL application increased C and N mineralization more than IF. As depth increased, more C and N mineralization occurred under CT due to plow layer mixing. Results indicated that long‐term tillage with PL application can increase soil C and N mineralization, nutrient retention, and organic matter.