Evidence suggests that immune dysfunction exerts a central role in the morbidity and mortality of sepsis. As the spleen is the largest lymphatic tissue in the body, its influence on immune regulation during sepsis should be explored. In this study, we analysed the immune alterations of the spleen of septic rats and the effects of splenectomy at 6 h, 12 h, and 24 h following caecal ligation and puncture (CLP). Results showed declines in CD4+ T cells and elevations in lymphocyte apoptosis, the percentage of Treg cells, and inflammatory cytokine levels (TNF-α, IL-6, and IL-10) in the spleens of CLP-induced septic rats. Moreover, splenectomy improved the survival of septic rats and bacterial clearance from peripheral blood. CLP-induced apoptosis of lymphocytes and the decreased CD4+ T cell percentage in the peripheral blood could be reversed in splenectomy-treated rats. Splenectomy greatly decreased the number of white blood cells, lymphocytes, monocytes, neutrophils, and serum concentration of TNF-α and IL-10 after CLP. Moreover, splenectomy alleviated pathologic damage to the liver and lungs and weakened expression of CD163. These novel findings demonstrate that immune disorders of the spleen are important pathogenic factors during the course of severe sepsis. Splenectomy could alleviate apoptosis and reduction of lymphocytes induced by sepsis, and lower the level of inflammation in the body. Reversing the immune suppression of the spleen may be a novel strategy to improve sepsis survival.