Abstract: Objective To investigate whether diosgenin (Dio) induces apoptosis in hepatocellular carcinoma (HCC) cells by regulating the mitochondrial fusion protein 2 (MFN2)-mediated mitochondrial autophagy pathway. Methods The expression of MFN2 in human normal liver cells (LO2) and HCC cells (HepG2) was detected by Western blot. MFN2 overexpression (Mfn2-OE) and knockdown (Mfn2-siRNA) plasmids were constructed and transfected into HepG2 cells. The effect of MFN2 on cell viability was analyzed by CCK-8 assay, Western blot, and flow cytometry. The regulatory effect of MFN2 on the PI3K/AKT signaling pathway was also explored. Cells were treated with different concentrations of Dio alone or with PINK1 knockdown, and the effect of Dio on MFN2, mitochondrial autophagy, and apoptosis was measured. Results The expression of MFN2 in HepG2 cells was significantly lower than that in LO2 cells. Overexpression of MFN2 markedly inhibited cell proliferation and induced apoptosis, while MFN2 knockdown promoted cell survival and suppressed apoptosis. Dio induced apoptosis in HepG2 cells in a concentration-dependent manner, upregulating MFN2, p-Parkin, and the LC3-Ⅱ/Ⅰ ratio, and downregulating p62 expression. Knockdown of MFN2 or PINK1 partially reversed the Dio-induced mitochondrial autophagy activation and apoptosis. MFN2 knockdown specifically activated the PI3K/AKT signaling pathway. Conclusions Diosgenin induces apoptosis in HCC cells through upregulation of MFN2, activating the PINK1/Parkin pathway-dependent mitochondrial autophagy. The absence of MFN2 may counteract these changes by activating the PI3K/AKT pathway.