In this study, we exposed the rats to 1.5 GHz (L10), 4.3 GHz (C10) or multi-frequency (LC10) microwave at an av-erage power density of 10 mW/cm2. Both single and multi-frequency microwave induced slight pathological changes in thymus and spleen. And, the white blood cells (WBCs) and lymphocytes in peripheral blood were decreased at 6 h and 7 d after exposure, suggesting immune suppressive responses were induced. Among lymphocytes, the B lymphocytes were increased while the T lymphocytes were decreased at 7 d after exposure in C10 and LC10 groups, but not in L10 group. Moreover, multi-frequency microwave regulated the B and T lymphocytes stronger than C band microwave. The results of proteomics showed that both single and mul-ti-frequency microwave regulated numerous genes associated with immune regulation and cel-lular metabolism in peripheral blood and spleen. However, multi-frequency microwave altered the expression of much more proteins. Moreover, multi-frequency microwave down-regulated T lymphocytes development, differentiation and activation associated proteins, while up-regulated B lymphocytes activation related proteins. In conclusion, multi-frequency mi-crowave of 1.5 GHz and 4.3 GHz produced immune suppressive responses via regulating im-mune regulation and cellular metabolism associated proteins. Our findings provided meaningful information for exploring potential mechanisms underlying multi-frequency induced immune suppression.