Soil carbon sequestration and potential sequestration are key issues in global carbon research, as the size of the soil organic carbon (SOC) stocks and their changes over time are important for understanding their role in climate change. In northern China’s agro-pastoral ecotone, we compared SOC data derived from 238 sampling sites during China’s National Soil Inventory in the 1980s with the results of field surveys at 644 sites in 2018 to calculate the changes in SOC stocks between the 1980s and 2018. We found that the mean SOC stock to a depth of 30 cm decreased significantly, from 4.48 kg C m⁻² in the 1980s to 3.60 kg C m⁻² in 2018, and the total SOC stock decreased from 2793.17 Tg to 2248.34 Tg. This amounted to an average decrease of 27.74 g C m⁻² yr⁻¹ over the 30-year study period. Furthermore, we used a land use and cover change transition matrix to quantify the amount of SOC change caused by these transitions; the SOC reduction could be mainly ascribed to overgrazing, grassland reclamation for agriculture, and grassland desertification. Large-scale ecological restoration projects have increased SOC reserves, but not enough to compensate for the loss of SOC caused by factors such as desertification and ecosystem degradation. We also performed regression analysis for the relationship between the change in the SOC stock and the rates of temperature and precipitation change. We found that increasing temperature non-significantly decreased SOC, whereas precipitation was significantly (P < 0.05) related to SOC change, but the R² was very low (0.01). Our analysis suggests that humans have affected SOC in northern China’s agro-pastoral ecotone more strongly than climatic factors.