Amino acids and peptides are important regulators of ecosystem functioning due to their potential role as direct nutrient sources for plants and soil microbes. However, the turnover and driving factors of these compounds in agricultural soils remain poorly understood. This study aimed to reveal the short-term fate of C-labeled alanine and tri-alanine derived C under flooding conditions of the top (0-20 cm) and sub-horizons (20-40 cm) of subtropical paddy soils taken from four long-term (31 years since treatment) nitrogen (N) fertilization regimes (i.e., without fertilization, NPK, NPK with straw return (NPKS) or with manure (NPKM)). Amino acid mineralization was strongly affected by the N fertilization regime and soil depth, while peptide mineralization was only distinct between soil layers. The average half-life of amino acid and peptide in the topsoil was 8 h across all treatments, which was higher than previously reported in uplands. The microbial turnover of amino acid and peptide was 7-10 times slower in the subsoil than in the topsoil, with a half-life of about 2-3 days. The half-life of amino acid and peptide for the respired pool was strongly associated with soil physicochemical characteristics, the total biomass, and the structure of soil microbial communities. The N fertilization regime and soil depth affected the substrate uptake rate by microorganisms, with greater uptake observed in the NPKS and NPKM treatments and the topsoil. Microbial amino acid uptake was correlated with the biomass of total and individual microbial groups, whereas microbial peptide uptake was associated with the soil microbial community structure and physicochemical characteristics. This suggests that there are various pathways of amino acid and peptide use by microorganisms under flooding conditions. We conclude that microbial mineralization of amino acid and its peptide in paddy soils under flooding conditions is slower than in upland soils, and that microbial uptake of these substrates is related to soil abiotic factors and the biomass and structure of soil microbial community. These findings have important implications for understanding nutrient cycling and ecosystem functioning in agricultural soils. [Abstract copyright: Copyright © 2023 Elsevier B.V. All rights reserved.]