Recalcitrant dissolved organic carbon (RDOC) represents a persistent fraction of the carbon pool and contributes substantially to regional carbon budgets in terrestrial aquatic ecosystems. Given the substantial carbon sequestration potential of karst waters, elucidating the dynamics and persistence of RDOC is essential for understanding carbon retention in these geologically distinctive environments. Karst reservoirs represent important yet highly complex components of regional carbon cycling. Here, RDOC accumulation and its underlying mechanisms were examined in the Dalongdong (DLD) Reservoir, a dissolved-carbon-rich karst water body, over three contrasting thermal phases: incubating thermal stratification (ITS), obvious thermal stratification (OTS), and mixing (MX). Our results revealed that RDOC dynamics were predominantly regulated by microbial processes rather than benthic carbon inputs. The establishment of thermal stratification generated pronounced physicochemical gradients that facilitated vertical niche partitioning among keystone taxa, thereby regulating DOM bioavailability through taxon-specific metabolic pathways. Bacterial network analysis further indicated that facilitative interactions favored the generation of labile carbon, whereas competitive interactions under environmental stress promoted RDOC accumulation. Notably, the distinctive geochemical conditions of the karst system facilitated Ca–P co-precipitation, resulting in persistent phosphorus limitation and an elevated C:P ratio in the water column. This nutrient imbalance reduced microbial carbon use efficiency and suppressed extracellular enzyme activities, consequently favoring the conversion of autochthonous labile carbon into more persistent RDOC. Collectively, these findings suggest that the synergistic coupling of the biological carbon pump (BCP) and microbial carbon pump (MCP), reinforced by geochemical phosphorus sequestration, represents an important mechanism underlying long-term carbon retention in alkaline, calcium-rich aquatic systems. Our findings further demonstrate that karst-specific geochemical conditions interact with thermal stratification to regulate microbial processes governing carbon persistence. In particular, MCP efficiency appears to be modulated by carbonate-weathering-derived dissolved inorganic carbon (DIC), providing mechanistic insights into the enhanced carbon sequestration capacity of geologically distinctive inland waters. Protecting these highly efficient carbon-sequestering ecosystems may therefore contribute to atmospheric CO₂ removal and provide an additional pathway for advancing global climate mitigation.