In order to study the spatial variability of soil organic matter and its main influencing factors, 193 soil profiles from the second soil survey in Guizhou province were used, based on the meteorological data, land use type map and soil type map of Guizhou province, geostatistics and multiple regression were used to analyze the explanatory ability of each factor to the spatial variability of topsoil organic matter. The results showed that: (1) The organic matter content of surface soil in Guizhou Province ranged from 0.05% to 36.68%, with an average of 5.23%±4.96% and a coefficient of variation of 94.90%. (2) The spatial distribution of topsoil organic matter was higher in the northeast and lower in the southwest. (3) According to the results of semi-variational function analysis, soil organic matter in Guizhou province was mainly affected by natural factors. The comprehensive explanatory ability of altitude, CEC, tillage and soil texture for the variation of organic matter content was 35.9%, and altitude, as the most important influencing factor, could independently explain 25.6% of the variation. CEC and farming methods could explain 11.3% and 7.4%, respectively. There was a negative correlation between surface soil organic matter and average annual temperature and a positive correlation between surface soil organic matter and altitude. There was a correlation between the distribution of surface soil organic matter and the distribution area proportion of carbonate rocks in the study area. On the whole, the distribution area proportion of karst increased with the influence of natural factors. The areas with 0~10% karst distribution area were mainly affected by human activities such as agricultural cultivation and fertilization, 11%~30% were mainly affected by altitude, 31%~50% were mainly affected by average annual temperature and CEC, and 51%~100% of the areas were more affected by elevation, average annual temperature, precipitation, CEC and pH. The results are helpful to understand quantitatively the spatial distribution of organic matter in Guizhou, and provide scientific basis and reference for soil fertility regulation and ecological management.