A bidirectional labeling method was established to distinguish the proportions of HCO3 - and CO2 utilization pathways of microalgae in Lake Hongfeng. The method was based on microalgae cultured in a medium by adding equal concentrations of NaH13CO3 with different d13C values simultaneously. The inorganic carbon sources were quantified according to the stable carbon isotope composition in the treated microalgae. The effects of extracellular carbonic anhydrase (CAex) on the HCO3 - and CO2 utilization pathways were distinguished using acetazolamide, a potent membrane-impermeable carbonic anhydrase inhibitor. The results show utilization of the added HCO3 - was only 8% of the total carbon sources in karst lake. The proportion of the HCO3 - utilization pathway was 52% of total inorganic carbon assimilation. Therefore, in the natural water of the karst area, the microalgae used less bicarbonate that preexisted in the aqueous medium than CO2 derived from the atmosphere. CAex increased the utilization of inorganic carbon from the atmosphere. The microalgae with CAex had greater carbon sequestration capacity in this karst area.