Hepatocellular carcinoma (HCC) remains a major clinical challenge. Although mitophagy is implicated in hepatocarcinogenesis, novel therapeutic options targeting mitophagy for HCC treatment still await further studies. Here, we demonstrate that sanguinarine induces cell death in HCC cell line MHCC-97H through the mitochondrial apoptosis pathway. Sanguinarine triggers mitochondrial dysfunction and PTEN-induced putative kinase 1 (PINK1)/Parkin upregulation and recruitment to mitochondria. Elevated levels of p62 and LC3-II/I ratios suggest that sanguinarine is both an inducer of autophagy and a blocker of autolysosome formation, which is further confirmed by LC3-II conversion levels in presence of autophagy and mitophagy inhibitors, as well as an autophagy activator. In addition, blocking autophagy promotes sanguinarine-induced cell death, indicating mitophagy plays a cytoprotective role in sanguinarine-treated cells. Our findings suggest that blocking mitophagy may contribute to sanguinarine-induced mitochondrial apoptosis through the prevention of damaged mitochondrial clearance.