Abstract: Liver cancer is one of the leading causes of cancer death in China, characterized by high incidence and poor traditional treatment outcomes. The marine lectin can mediate intercellular interactions, induce apoptosis, regulate cytokine expression, and play a significant role in anti-tumor therapy. Oncolytic virus therapy through direct oncolysis and induction of immune responses, offers promise for cancer treatment. The oncolytic vaccinia virus carrying Aphrocallistes vastua lectin (AVL) (oncoVV-AVL) has demonstrated efficacy against tumors, however, its underlying mechanisms require further investigation. In this study, we used flow cytometry and Western blotting to explore its mechanism of inducing mitophagy, thereby inhibiting the proliferation of Hep3B hepatocellular carcinoma cells. Results indicate that oncoVV-AVL promotes mitochondrial reactive oxygen species levels, reduces the mitochondrial membrane potential, leading to mitochondrial depolarization and damage, consequently activating the PINK1-Parkin signaling pathway, and ultimately improving anti-cancer efficacy through mitochondrial autophagy. This study provides new insights into gene therapy for cancer treatment and enriched the application pathways of marine biological resources.