In recent years, an increasing number of ecosystems are threatened by seasonal flooding, changing non‐flooded shoreland (NF) into seasonally flooded wetland (SF), but the consequences of this hydrological change for soil organic carbon (SOC) dynamics remain unknown. In this study, we investigated how the SOC content was determined by flooding duration and soil physicochemical variables in adjacent SF and NF at six depths (0–10 cm, 10–20 cm, 20–30 cm, 30–50 cm, 50–70 cm, and 70–100 cm) at Shengjin Lake in subtropical China. Soil physicochemistry and SOC composition were analyzed, and Fourier‐transformed infrared spectroscopy (FTIR) was used to resolve the SOC composition. Neither SOC content nor the vertical distribution of SOC was distinguishable between the sites. However, FTIR data revealed that plant‐originated aliphatics and amides were higher at NF than SF sites, with the opposite pattern for aromatics. At SF sites, SOC content was positively affected by soil moisture and flooding duration and was negatively impacted by soil particle size at most soil layers. At NF sites, SOC content was mainly affected by silt and total Fe at the top 20 cm soil, while a higher fraction of plant‐derived labile C was positively correlated to SOC contents at 30–100 cm depth. The results hence indicated a strong effect of seasonal flooding on SOC dynamics in terrestrial ecosystems. SOC stabilization induced by low mineralization and high adsorption played a central role at SF sites, while SOC formation through plant input was more important at NF sites. Our findings suggest that management strategies designed to conserve SOC will need to be site‐specific.