Using the grid‐based sampling method, 2755 soil profiles consisting of 22,057 soil samples were sampled and analyzed to study the spatial distribution and storage of soil organic carbon (SOC) in the Houzhai River Basin in Guizhou Province, a typical karst region in southwestern China. The results suggest that the total levels ofSOCstored in the 0–10, 0–20, 0–30, and 0–100 cm soil layers of the Houzhai River Basin are approximately 1.48 × 10⁸, 2.65 × 10⁸, 3.44 × 10⁸, and 5.39 × 10⁸ kg C, respectively.SOCcontent and soil bulk density levels were found to vary depending on the land use. In the 0–50 cm soil layer, forestSOCcontent levels (including arbor forests, arbor–shrub mixed forests, shrubland, and shrub grassland) are generally higher than those of uncultivated land, followed by grassland and cropland (including paddy fields, arid land, sloping cropland, and abandoned cropland), gardens, and orchard. It is believed that land use is a vital factor directly affecting carbon storage patterns in the study area. Soil thickness, rock outcrop, and slope gradients are the main factors in arrangement of land use by local governments and peasants. This study has established a feasible method for assessment ofSOCstock in karst mountainous areas. The findings of this study are applicable to other karst mountainous regions globally.