This study aims to investigate the immunomodulatory effect of Momordica charantia polysaccharide (MCP) on mice spleen lymphocytes and its potential mechanism at the cellular and molecular levels. Compared with the blank group and levamisole group, mice spleen lymphocytes are stimulated by MCP with different concentrations (40, 80, 160, 320 μg/mL) in vitro. The effects of MCP on the proliferation of mice spleen lymphocytes, phagocytic activity of peritoneal macrophages, secretion and mRNA expression of IL-4, IFN-γ, IL-6 and IL-12 in lymphocyte supernatant are examined. The results show that compared with the blank group, the cell proliferation index increases exponentially as the concentration (P<0.05) raises, and MCP promotes cell proliferation in different degrees. MCP enhances phagocytosis of macrophages in different degrees (P<0.05). Compared with the blank group, the secretion of cytokines IL-4, IFN-γ, IL-6 and IL-12 all increase, with the largest increase being 42.73%, 18%, 27.81% and 29.42%, respectively. The mRNA expressions of IL-4, IFN-γ, IL-6 and IL-12 increase significantly, and the highest expression levels are 9.18 times, 7.53 times, 9.37 times and 8.95 times of the blank group, respectively, with significant difference (P<0.05). It is understood that MCP can effectively improve the secretion and mRNA expression of cytokines IL-4, IFN-γ, IL-6 and IL-12, and promote cell proliferation, enhance phagocytic activity of macrophages, thus improving the immune function of the body.