Separation of mitogenic fraction (PH-I) from Pseudostellaria heterophylla and characterization of its biological activities were investigated. PH-I was isolated as an alcohol-insoluble fraction from the hot water extract obtained by heating the roots of P. heterophylla in water at 80°C for 4 h. It is a water-soluble substance consisting of mainly carbohydrates (56.8%) and a small amount of proteins (7.6%). The incorporation of tritiated thymidine into the DNA of murine spleen lymphocytes could be stimulated by PH-I in a dose-dependent manner. PH-I could act as a priming agent for the release of tumor necrosis factor (TNF) in mice. Moreover, PH-I exhibited potent anti-tumor activities against Ehrlich ascites tumor (EAT) cells in vivo but not in vitro.