Improved understanding of long-term tillage management on soil C and N dynamics is important for sustaining soil productivity. The objectives of this study were to determine effects of long-term tillage practices (9-yr) on seasonal and profile changes in soil microbial biomass C (SMBC) and N (SMBN), and mineralizable C and N in continuous cotton (Grossypium hirsutum L.) under no-tillage (NT), minimum tillage (MT) and conventional tillage (CN) under zero, low (20 lb N A-1) and high (60 lb N A-1) N fertilization. Additional measurements included soil bulk density and gravimetric water content. A Victoria clay (fine hyperthermic montmorillonitic Pellusterts) was sampled at three soil depths (0-2, 2-5 and 5-8 inch) before planting in March, during flowering in May, and after cotton harvest in August. No-tillage treatment tended to have higher soil bulk density and more surface water content with and without fertilization. Soil in the CN system showed the lowest water content. No-tillage also exhibited higher inorganic N concentration at planting than CN and MT. Inorganic N increased with the increase in fertilizer rates for all tillage systems but declined through the season. Microbial biomass C was greatest under MT at 0 to 8 inch depth as the season started because of residue accumulation and incorporation. No-tillage, however, had higher ability to maintain microbial biomass as season progressed and kept more biomass than CN and MT at flowering and harvest. The decrease in labial C quality and availability tended to decrease C and N mineralization potential through the season. Fertilization, however, caused increases in mineralizable C and N for all tillage treatments while NT with highest substrate showed greatest mineralization.