Abstract Grassland ecosystems play an essential role in climate regulation through carbon (C) storage in plant and soil. But, anthropogenic practices such as livestock grazing, grazing related excreta nitrogen (N) deposition, and manure/fertilizer N application have the potential to reduce the effectiveness of grassland C sink through increased nitrous oxide (N2O) and methane (CH4) emissions. Although the effect of anthropogenic activities on net greenhouse gas (GHG) fluxes in grassland ecosystems have been investigated at local to regional scales, estimates of net GHG balance at the global scale remains uncertain. With the data-model framework integrating empirical estimates of livestock CH4 emissions with process-based modeling estimates of land CO2, N2O and CH4 fluxes, we examined the overall global warming potential (GWP) of grassland ecosystems during 1961–2010. We then quantified the grassland-specific and regional variations to identify hotspots of GHG fluxes. Our results show that, over a 100-year time horizon, grassland ecosystems sequestered a cumulative total of 113.9 Pg CO2-eq in plant and soil, but then released 91.9 Pg CO2-eq to the atmosphere, offsetting 81% of the net CO2 sink. We also found large grassland-specific variations in net GHG fluxes, with pasturelands acting as a small GHG source of 1.52 ± 143 Tg CO2-eq yr−1 (mean ± 1.0 s.d.) and rangelands a strong GHG sink (−442 ± 266 Tg CO2-eq yr−1) during 1961–2010. Regionally, Europe acted as a GHG source of 23 ± 10 Tg CO2-eq yr−1, while other regions (i.e. Africa, Southern Asia) were strong GHG sinks during 2001–2010. Our study highlights the importance of considering regional and grassland-specific differences in GHG fluxes for guiding future management and climate mitigation strategies in global grasslands.