Objective To explore the differentiation of HepG2 cells induced by mouse embryonic hepatocytes at day 13. 5 of gestation through the inhibition of β-Catenin. Methods The distribution of hepatocyte marker molecule- albumin (ALB) was detected by immunofluorescence to identify the hepatocytes of mouse embryo on day 13. 5. After HepG2 cells were co-cultured with embryonic hepatocytes at day 13. 5 of gestation for 24 h and 48 h, the mRNA expressions of AFP (biomarker of hepatocellular carcinoma), HNF-4α (controller of hepatocyte differentiation), CYP1B1 (biomarker of mature hepatocytes) and ADH1C ( another biomarker of mature hepatocytes) were measured by reverse transcriptional quantitative polymerase chain reaction (RT-qPCR). After HepG2 cells were co-cultured with day 13. 5 of gestation embryonic hepatocytes for 48 h, the morphology was observed; AFP, HNF-4α and β-catenin expressions were measured by Western blot and β-Catenin distribution was determined by immunofluorescence. Following treatment with the β-catenin inhibitor, morphology was observed and AFP content was measured. Results Most cells were ALB-positive. The relative mRNA expression of AFP decreased significantly after 24 h and 48 h co-culture, and HNF-4α, CYP1B1 and ADH1C increased significantly at 24 h and 48 h co-culture compared with the levels in the controls. The proliferation of HepG2 cells was suppressed and cell morphology tended to be hexagonal like normal hepatocytes; AFP protein content decreased and HNF-4α protein content increased after co-culture with embryonic hepatocytes at day 13. 5 of gestation for 48 h. Compared with the levels in the control, β-Catenin protein content decreased in the co-culture group and β-catenin was markedly attenuated in the co-cultured HepG2 cells in immunofluorescence result. Compared with the effects in the control group, β- catenin inhibitor treatment caused notable morphological changes of HepG2 cells and induced more dramatic morphological changes of HepG2 cells; furthermore, it reduced AFP protein content. Conclusions HepG2 cells may be induced to undergo differentiation by embryonic hepatocytes at day 13. 5 of gestation mainly through the suppression of β-catenin.