Matrine, one of the main components extracted from Sophora flavescens, has exhibited pharmacological effects on the differentiation in rat liver oval cells. However, its function and mechanism have not yet been fully elucidated. To further investigate them, an in vitro model was established using a rat liver oval cell line called WB-F344 and treated with matrine. Initially, a significant increase in the number of monodansylcadaverine-positive cells and in the levels of microtubule-associated protein 1A/1B-light chain 3 (LC3)-II, which is a specific marker for detecting autophagy, was observed in matrine-treated cells. This indicated that autophagy was stimulated by matrine, which was further confirmed by transmission electron microscopy. Additionally, the apoptotic oval cells were easily detected under matrine treatment using an Annexin-V-fluorescein isothiocyanate/propidium iodide assay, indicating that autophagy and apoptosis were synchronously induced by matrine. A decrease in B-cell lymphoma (Bcl-2) mRNA expression, but an increase in Bcl2-associated X protein (Bax) mRNA expression were observed in matrine-treated cells, which led to an upregulation of the Bax/Bcl-2 ratio, a molecular marker for determining the extent of apoptosis. Next, the molecular mechanism of matrine-induced autophagy and apoptosis was analyzed in WB-F344 cells. beta-catenin degradation was downregulated by matrine and rapamycin, a foregone chemical agonist of autophagy, whereas it was upregulated by 3-methyladenine, a specific inhibitor of autophagy. Additionally, beta-catenin activation induced an increase in LC3-II levels and reversed the Bax/Bcl-2 mRNA ratio under matrine treatment, whereas inhibition of beta-catenin by RNA interference induced a decrease of the LC3-II amount and of the Bax/Bcl-2 mRNA ratio. Finally, matrine treatment attenuated p53; however, with little or no change in LC3-II levels, but a decrease in beta-catenin levels occurred in WB-F344 cells upon treatment with pifithrin-alpha, a chemical inhibitor of p53, revealing that p53, interfering with beta-catenin, may not be involved in matrine-induced autophagy in WB-F344 cells. These results demonstrate that beta-catenin is involved in matrine-induced autophagy and apoptosis in WB-F344 cells, while beta-catenin is negatively regulated by autophagy. and positively by p53, indicating that beta-catenin may be involved in the crosstalk between autophagy and apoptosis in WB-F344 cells.