Nitrogen (N) is the prime nutrient for crop production and carbon-based functions associated with soil quality. The objective of our study (2012 to 2019) was to evaluate the impact of variable rates of N fertilization on soil organic carbon (C) pools and their stocks, stratification, and lability in subtropical wheat (Triticum aestivum)-mungbean (Vigna radiata)-rice (Oryza sativa L) agroecosystems. The field experiment was conducted in a randomized complete block design (RCB) with N fertilization at 60, 80, 100, 120, and 140% of the recommended rates of wheat (100 kg/ha), mungbean (20 kg/ha), and rice (80 kg/ha), respectively. Composite soils were collected at 0-15 and 15-30 cm depths from each replicated plot and analyzed for microbial biomass (MBC), basal respiration (BR), total organic C (TOC), particulate organic C (POC), permanganate oxidizable C (POXC), carbon lability indices, and stratification. N fertilization (120 and 140%) significantly increased the POC at both depths; however, the effect was more pronounced in the surface layer. Moreover, N fertilization (at 120% and 140%) significantly increased the TOC and labile C pools when compared to the control (100%) and the lower rates (60 and 80%). N fertilization significantly increased MBC, C pool (CPI), lability (CL.sub.I ), and management indices (CMI), indicating improved and efficient soil biological activities in such systems. The MBC and POC stocks were significantly higher with higher rates of N fertilization (120% and 140%) than the control. Likewise, higher rates of N fertilization significantly increased the stocks of labile C pools. Equally, the stratification values for POC, MBC, and POXC show evidence of improved soil quality because of optimum N fertilization (120-140%) to maintain and/or improve soil quality under rice-based systems in subtropical climates.