Soil organic carbon (SOC) dynamics along elevational gradients are highly uncertain due to our limited knowledge of the underlying mechanisms that determine SOC release. By combining field investigation and laboratory aerobic incubation with multiple soil analytical approaches, we explored the determinants of SOC release along an elevational gradient (3200–4200 m) in the Tibetan alpine grassland. We illustrated that the SOC quantity (i.e., the initial standing SOC stock) and SOC quality (i.e., chemical recalcitrance, physico-chemical protection and biological recalcitrant fraction) increased gradually with elevation, reaching a maximum value at the middle altitude (∼3600 m), while an opposite unimodal distribution pattern was observed in CO2–C release. Our results also showed that SOC quality explained more variance in CO2–C release than did SOC quantity and soil properties. The proportion of stable SOC fractions in larger initial SOC stocks is higher, especially the recalcitrant SOC pool and mineral-associated OC fractions, which could be the potential mechanism behind high initial SOC stocks leading to lower soil CO2–C release. Collectively, our results provide compelling evidence that SOC quality plays a primary role in the elevational pattern of CO2–C release in Tibetan alpine grassland. Our findings highlight the importance of SOC quality in predicting SOC dynamics in the Tibetan alpine grassland under climate change scenarios.