Soil organic carbon represents a major carbon pool in terrestrial ecosystems. Currently, in karst areas, there is considerable uncertainty regarding the estimation of soil organic carbon pools due to limited profile numbers and heterogeneity. Using a grid-based sampling method, 2755 soil profiles consisting of 22,057 soil samples were analyzed to determine the spatial distribution and storage of soil organic carbon (SOC) in the Houzhai River Basin in the Guizhou Province in southwest China. The results suggest that the total SOC storages in the 0–20 and 0–100 cm soil horizons of the Houzhai River Basin are approximately 2.65 × 10⁸ and 5.39 × 10⁸ kg C, respectively. The differences in the soil bulk density between different soil types were considerable. The average soil bulk density of large mud field loam, which ranged from 1.25 to 1.52 g/cm³, was significantly higher than that of the other soil types. The differences in soil organic carbon content were obvious as well. As soil depth increased, the organic carbon content gradually decreased. At a depth of 0–50 cm, the soil organic carbon content significantly decreased. In contrast, soil organic carbon content decreased slowly from 50 to 100 cm, indicating that a depth of 50 cm is the turning point for organic carbon content. The different degrees of soil organic carbon storage in different soil types were mainly caused by differences in organic carbon density. In karst regions, the soil parent material limits the spatial distribution of soil organic carbon, and soluble rock is the base development material. Soil thickness, slope gradient, and rock exposure are the primary factors leading to differences in SOC storage in different soil types. In addition, this study also suggests that increased attention should be paid to rock exposure in the assessment of SOC stocks in karst mountainous areas, such as the Houzhai River Basin.