Arginase I has been documented to impair T cell function and attenuate cellular immunity, however, there is little evidence to reveal the effect of arginase I on macrophage function. Recently, recombinant human arginase I (rhArg) has been developed for cancer therapy and is in clinical trial for hepatocellular carcinoma, whereas the potential immunosuppression induced by rhArg limited its therapeutic efficacy. To improve the clinical outcome of rhArg, addressing the immune suppression appears to be particularly important. In this study, we found that rhArg attenuated macrophage functions, including inhibiting macrophage cell proliferation, nitric oxide (NO) and reactive oxygen species (ROS) production, cytokine secretion, MHC-II surface expression, and phagocytosis, thereby inducing immunosuppression in lipopolysaccharides (LPS)/interferon- (IFN-)-activated macrophages. Notably, we observed that rhArg downregulated autophagy in activated macrophages. Moreover, application of trehalose (an autophagy inducer) significantly restored the impaired immune function in activated macrophages, suggesting the essential role of autophagy in rhArg-induced immunosuppression. To further illustrate the effect of autophagy in immunosuppression, we then observed the effect of 3-MA (an autophagy inhibitor) on the immune function of macrophages. As expected, inhibiting autophagy by 3-MA attenuated immune functions in activated macrophages. Collectively, this study elucidated that rhArg induced immunosuppression in activated macrophages via inhibiting autophagy, providing potential strategy to ameliorate the immune suppression which is of great significance to cancer therapy and facilitating the development of rhArg as a potential therapy for malignant carcinomas.