Effects of human activity on ecosystem carbon fluxes (e.g., net ecosystem exchange (NEE), ecosystem respiration (R eco), and gross ecosystem exchange (GEE)) are crucial for projecting future uptake of CO2 in terrestrial ecosystems. However, how ecosystem that carbon fluxes respond to grazing exclusion is still under debate. In this study, a field experiment was conducted to study the effects of grazing exclusion on R eco, NEE, and GEE with three treatments (free-range grazing (FG) and grazing exclusion for 3 and 5 years (GE3 and GE5, respectively)) in a meadow grassland on the Tibetan Plateau. Our results show that grazing exclusion significantly increased NEE by 47.37 and 15.84 %, and R eco by 33.14 and 4.29 % under GE3 and GE5 plots, respectively, although carbon sinks occurred in all plots during the growing season, with values of 192.11, 283.12, and 222.54 g C m−2 for FG, GE3, and GE5, respectively. Interestingly, grazing exclusion increased temperature sensitivity (Q 10) of R eco with larger increases at the beginning and end of growing season (i.e., May and October, respectively). Soil temperature and soil moisture were key factors on controlling the diurnal and seasonal variations of R eco, NEE, and GEE, with soil temperature having a stronger influence. Therefore, the combined effects of grazing and temperature suggest that grazing should be taken into consideration in assessing global warming effects on grassland ecosystem CO2 exchange.