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Lienal peptide (LP), extracted from the spleen of healthy calves, has been reported to be a unique immune modulator which shows notable immunomodulatory effects on immune system function. This study was designed to further investigate the immunomodulatory effect of LP in vitro and in vivo. The results showed that LP significantly decreased the LPS-induced overexpression levels of pro-inflammatory cytokines including IL-1β, IL-15, TNF-α and MIP-1α, through the NF-κB pathway. Moreover, LP showed an immunologic enhancement effect on immunosuppressed mice which were induced by cytarabine. The percentage of activated cells for bone marrow B lymphocytes, spleen lymphocytes, NK cells and peritoneal macrophages were notably increased after LP treatment. Furthermore, the administration of LP significantly reduced DNFB-induced inflammatory cell infiltration and restored CFA-induced arthritis in rats as evidenced by decrease in paw swelling and regulation of cytokines balance in serum. In conclusion, LP has outstanding immunomodulatory activity and could be served as a potential candidate for the management of patients with immune system disorders.The effects of lienal peptide on immune system were evaluated in vivo and in vitro.Lienal peptide shows dual regulatory function on immune system.Lienal peptide may serve as a potential candidate for patients with immune system disorders.