Carbonate rock weathering coupled with aquatic photosynthesis in karst areas is an important part in the formation of terrestrial carbon sinks. The capacity of photosynthetic carbon sequestration by aquatic microalgae and carbonic anhydrase (CA) is integral in the estimation of carbon sink potential of karst aquatic ecosystems. To date, carbon sequestration by aquatic microalgae in karst areas has been investigated in laboratory experiments. In the present work, the capacity of carbon sequestration by microalgae and CA under natural karst aquatic conditions and the main environmental factors were investigated in field in-situ mesocosms. The Sizhitan Pond of the Huixian karst wetland in Guilin City, Guangxi Province, China, was selected as a typical karst natural water body for this study. The capacity of photosynthetic carbon sequestration varied with microalgal community composition. The microalgal communities with active extracellular CA showed high capacity of carbon sequestration. The average conversion of inorganic carbon to relatively stable organic carbon by microalgae in the Huixian karst wetland aquatic ecosystem was estimated as 4207.5 t C/a. Approximately 28.7% of the bicarbonate fed by the karst underground river was fixed into organic carbon by microalgal photosynthesis. The major environmental factors affecting the capacity of carbon sequestration by microalgae in the karst wetland aquatic ecosystem were the water CA activity, illumination, temperature, total phosphorus and total nitrogen. This study is the first to address the contribution of aquatic microalgae and CA to carbon sequestration under natural karst aquatic conditions. The findings contribute to establishing groundwork for substantiating the carbon sink potential in global karst ecosystems.