<p>How the intensity of biological carbon pump (BCP) regulates the accumulation of autochthonous organic carbon (AOC) and the stabilization of recalcitrant organic carbon (ROC) in reservoir sediments remains poorly understood. Here, we integrated stable carbon and nitrogen isotopes (δ¹³C and δ¹⁵N), organic carbon (OC) classification, and molecular fingerprinting to elucidate the sources and stabilization mechanisms of sedimentary OC in the Songbaishan Reservoir. Thermal stratification enhanced the BCP, resulting in a drawdown of dissolved inorganic carbon by 9.1 mg L⁻¹ and concurrent enrichment of δ¹³C_DIC by + 2.7‰ relative to inflowing rivers. This shift, driven by distinct nutrient (carbon-phosphorus) limitations, promoted substantial AOC production. The AOC accounted for 65.8 ± 12.1% of the sedimentary OC pool, supporting a high organic carbon burial rate (OCBR) of 89.5 g C m⁻² a⁻¹. Additionally, ROC constituted 59.8% of the sedimentary OC, indicating its dominant role in carbon sequestration. The enhanced OCBR in karst reservoirs is therefore primarily driven by the accumulation of sedimentary ROC, which constitutes the key mechanism ensuring efficient carbon sequestration. Consequently, we propose the ratio of ROC to total organic carbon as an effective and practical metric for evaluating a reservoir’s carbon sequestration capacity.</p> Graphical Abstract <p></p>