BackgroundGrazing exclusion is a practical approach to restore vegetation in degraded grasslands and enhance soil organic carbon (SOC) sequestration. However, the dynamics and drivers of SOC in grasslands after grazing exclusion have not been well documented, especially in ecosystems with cold climates.MethodsHere, we established 14 paired treatments (grazing exclusion vs. free-grazing) along a 600-km transect in the northeastern zone of the Qinghai-Tibet Plateau. After six years, we analyzed vegetation biomass dynamics and measured the soil physicochemical properties and organic C concentration across three depths (0–10, 10–20, and 20–30 cm).ResultsGrazing exclusion significantly increased above- and belowground biomass (139.85% and 43.30%, respectively), pH (1.38%), total phosphorus (3.29%), nitrate nitrogen (18.03%), and ammonium nitrogen (17.81%), but significantly decreased soil bulk density (2.43%) and clay content (10.49%), particularly in 0–30 cm. Specifically, SOC concentrations positively responded to grazing exclusion (0–10 cm) in 9 of the 14 sites evaluated. The effects of grazing exclusion on SOC concentrations were significantly higher in areas with a mean annual temperature (MAT) below 0 °C compared to those in sites with a high MAT (> 0 °C). The SOC concentrations significantly correlated with the mean annual precipitation (MAP) in both treatments, but these correlations diminished with increasing soil depth. Ridge regression analysis showed that soil chemical properties (e.g., total nitrogen and phosphorus) positively influenced SOC accumulation, while MAT negatively influenced it after grazing exclusion. Path analysis further revealed that MAT indirectly regulated SOC dynamics via soil chemical properties.ConclusionsOur study highlights that grazing exclusion results in an asynchronous SOC and plant biomass accumulation and may be more beneficial for SOC sequestration in Qinghai-Tibet Plateau grasslands with lower temperatures. Also, humid climates promote SOC concentration in alpine grasslands. These results could help develop management practices and policies that promote sustainable grassland management.