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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 (CLI), 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. [ABSTRACT FROM AUTHOR]