Matrine, one of the main active components extracted from dry roots of Sophora flavescens Ait (Leguminosae), has been reported to have anticancer effects on a number of cancer cell lines, but the anticancer mechanism of matrine remains elusive. This study shows that matrine also displays anticancer activity on human hepatocellular carcinoma (HepG2) cells. In this work, the optimal cultivation condition for HepG2 cells was determined using the combinatorial orthogonal test design [L18 (21 x 37)]. Exposure of HepG2 cells to matrine resulted in inhibition of proliferation in both a time- and dose-dependent manner, as measured by morphology observation, hematoxylin and eosin (H&E) staining, and MTT assay (p<0.05). Further immunohistochemical analyses revealed that the expression of alpha fetal protein (AFP), proliferating cell nuclear antigen (PCNA), C-myc and Bcl-2 was down-regulated significantly, but the expression of Bax was up-regulated higher than untreated cells. The results demonstrated that matrine inhibited HepG2 cells proliferation primarily via up-regulating or down-regulating expression of the tumor relevant proteins.