Effects of heavy grazing on soil nitrogen mineralization and temperature sensitivity in the Eastern Eurasian Steppe were studied using laboratory incubation methods by comparing non-grazing plots with heavy grazing plots at different sites. The results showed that accumulations of soil nitrogen and soil nitrogen mineralization rates were significantly higher in sites with a higher aridity index (AI) than sites with a lower AI (P < 0.05), which indicated that soil nitrogen turnover rates were high in relatively humid sites. In sites with a higher AI, the amount and rate of soil ammonium nitrogen reduction in heavy grazing plots were lower than that of non-grazing plots (P < 0.05), and heavy grazing significantly decreased accumulations of soil nitrate nitrogen (A_nit), accumulations of soil inorganic nitrogen (A_min), soil nitrification rate (R_nit), and net nitrogen mineralization rate (R_min) (P < 0.05). In sites with lower AI, the amount and rate of soil ammonium nitrogen reduction in heavy grazing plots were higher than that of non-grazing plots (P < 0.05), and heavy grazing had little effect on A_nit, A_min, R_nit, and R_min (P>0.05). The temperature sensitivity of net nitrogen mineralization (Q₁₀) ranged from 1.61 to 2.06 and heavy grazing had little effect on Q₁₀. Q₁₀increased with increasing latitude and showed a significant negative correlation with the substrate quality index (P < 0.05). There was a significant negative correlation between substrate quality index and apparent activation energy (P < 0.05). Furthermore, the response ratios of A_nit, A_min, R_nit, and R_min to heavy grazing were significantly negatively correlated with AI (P < 0.01). Our results emphasized that the effects of heavy grazing on soil nitrogen mineralization in the Eastern Eurasian Steppe was regulated by climate (temperature and precipitation).