The potential effects of microwave radiation have gained increasing attention due to its widespread application in daily life. This study aimed to investigate its effects on rat immune function. Male Wistar rats were subjected to 2.856 GHz microwave radiation for 20 min. Cytokine levels in serum were measured by ELISA at 0 and 7 days' post-exposure. HE staining was used to observe the structure of the spleen. Morphology and proliferation of spleen mixed lymphocytes were detected after 24 h of culture, and proliferation was evaluated by the CCK-8 assay. Expression of HSP70 and calreticulin (CRT) was assessed by WB. Rats showed an increased secretion of inflammatory factors in serum. Spleen tissue revealed no significant damage. Compared to the control group, irradiated groups (R-0d and R-7d) showed a significant increase and thickening of the white pulp. The boundary between the red and white medulla in the R-7d group appeared blurred. CCK-8 assay showed that splenic mixed lymphocytes in the irradiated group showed a significant proliferation. There was no significant difference in HSP70 and CRT expression between the exposed and control groups. Microwave exposure might activate the immune system of rats, potentially triggering a T cell-mediated inflammatory response.