Due to specific soil properties and management practices, soils in vineyards are sensitive to degradation. The aims of this study were to examine (i) the state of soil organic carbon (SOC) in vineyards compared to other agricultural land, (ii) the influence of different fertilization strategies and soil type on SOC content and (iii) the rate of SOC change over time and potential of deep tillage for SOC preservation in subsoil. The study was carried out at 16 representative vineyard locations of the Tri Morave region, which represents the largest vine growing region in Serbia. The analyzed area included 56 vineyard plots. Results showed that SOC stocks in the topsoil and subsoil were lower than the average for agricultural land in Serbia. The soil type was an important predictor of carbon storage in the topsoil. An adequate application of inorganic fertilizers or green manure combined with farmyard manure initially resulted in the highest SOC contents. Continuous application of inorganic fertilizer without organic amendments has led to a decrease of SOC in topsoil. High rates of SOC stock change in topsoil accompanied a rapid reduction of SOC in the earlier stage of cultivation. In all investigated subsoils, SOC increased, except for unfertilized vineyards. Deep tillage has the potential to preserve SOC in the deeper soil layer and prevent carbon loss from the topsoil. More attention should be paid to the soil conservation practices to meet environmental sustainability of viticulture.