Objective The spatial and temporal distribution pattern and evolution characteristics of ecosystem carbon storage, its response to land use change, and the vulnerability of ecosystem carbon storage service in each county of Guizhou Province were analyzed in order to provide a scientific basis and reference for regional land use management decision-making and to realize the "dual carbon" goal. Methods The InVEST model and the potential impact (PI) index were used to analyze the characteristics of carbon stock changes and the vulnerability of ecosystem carbon storage services in Guizhou Province from 2000 to 2020. Results ① Land use type in Guizhou Province changed significantly during the last 20 years. 14.10% and 17.29% of the land changed type in the first 10 years and the last 10 years, respectively. Cultivated land was the main source of construction land expansion. ② Ecosystem carbon storage in Guizhou Province decreased by 0.24 billion tons in the past 20 years. Forest land reductions and construction land expansion were the main reasons for carbon storage reduction. ③ All Moran's I indices of ecosystem carbon stocks in Guizhou Province exceeded 0, indicating significant positive spatial correlation and agglomeration. The hot spot/cold-spot analysis showed that the distribution of carbon stock hot spots was relatively decentralized while the distribution of cold spots was centralized and stable. ④ The PI indices for Guizhou Province in the first and second 10-year periods were -1.27 and -0.15, respectively, indicating an improvement in vulnerability. There were spatial differences in vulnerability among counties, with significant negative effects in the peripheral counties. The vulnerability of 81.82% of the counties decreased in 20 years. Conclusion The transfer of forest land and the expansion of construction land in Guizhou Province had a significant impact on carbon stocks and the vulnerability of carbon stock services. In the future, land use structure should be optimized, and planning and management should be strengthened.