Objective To induce human gallbladder epithelial cells (hGBECs) differentiating into hepatocyte-like cells through adding small molecule compounds which could change cell fate. Methods The primary hGBECs were three-dimensionally cultured in Matrigel by adding B27 supplement, N2 supplement, N-acetyl-L-cysteine, epidermal growth factor and hepatocyte growth factor in the medium. Small molecule compounds/proteins were added to induce cell differentiation, and the key molecules were screened out. Polymerase chain reaction (PCR), quantitative PCR, and immunofluorescence staining were used to detect the expression of stem cell markers and liver cell related markers. The liver function was evaluated by lipid BODIPY-493 staining, glycogen periodic acid-Schiff staining, and albumin enzyme-linked immunosorbent assay. Results Three-dimensionally cultured hGBECs expressed hepatic stem/progenitor cells-related markers, such as hematopoietic stem cell antigen CD133, epithelial cell adhesion molecule, and hepatocyte nuclear factor 4α. Transforming growth factor β (TGF-β) and Notch signaling pathway inhibitors were the key molecules for inducing hGBEC differentiation. The cells obtained under differentiation conditions expressed liver cell functional markers α1-antitrypsin, cytochrome P450 3A4, albumin and fumarate acetate hydrolase. The cells could store glycogen, synthesize fat and secrete albumin. Conclusion hGBECs can be cultured in vitro for a long time, and hGBECs can be induced to differentiate into hepatocyte-like cells with partial liver function by inhibiting TGF-β and Notch signaling pathways.