Soils hold the largest carbon stock in terrestrial ecosystems. Soil organic carbon (SOC) is formed under a combination of bioclimatic and land-use conditions. Therefore, one would expect changes in SOC stocks with land use changes like urbanization. So far, the majority of regional studies on SOC stocks exclude urban areas. The urban environment has a unique set of specific features and processes (e.g., soil sealing, functional zoning, settlement history) that influence SOC stocks and its spatial variability. This study aims to improve our understanding of urban SOC in comparison with agricultural and natural areas for the Moscow region (Russia). SOC content was studied in different land use types, soils, and urban zones through stratified random sampling. Samples of topsoil (0–10 cm) and subsoil (10–150 cm) were taken at 155 locations. SOC contents were significantly higher in urban areas compared with non-urban areas (3.3 over 2.7%). Further analyses proved that the difference can be explained by the so-called “cultural layer”, which is the result of human residential activity and settlement history. SOC contents in the urban environment presented a very high spatial heterogeneity with standard deviations of urban SOC considerably higher than those for agricultural and natural areas. Soil depth, soil type and land-use factors had a significant influence on SOC variability determining more than 30% of the total variance. SOC contents in urban topsoil were mostly determined by soil type. In natural and agricultural areas soil type and land-use determined SOC contents. The results confirm the unique character of urban SOC and the need to reconsider established scientific and management views on regional SOC assessment, taking into account the role of urban carbon stocks.