Hepatocellular carcinoma (HCC) is a primary malignancy arising from hepatocytes. In the clinical management of HCC, suboptimal therapeutic responses are frequently observed, which are closely associated with reduced overall survival rates. Pharmacological modulation of autophagic pathways represents a promising therapeutic strategy to resensitize tumor cells to targeted agents. Lasiodin (LAS) is a diterpenoid with antitumor and anti-inflammatory activities. However, the role of LAS in autophagy in HCC cells has not been investigated.To elucidate the specific mechanism by which LAS inhibits autophagy in HCC cells and investigate its impact on the sensitivity of HCC cells to sorafenib.We evaluated the effects of LAS on the proliferation and growth of HCC cells through Cell Counting Kit-8 (CCK-8) assay, apoptosis assay, and cell-cycle analyses. To investigate the specific molecular mechanisms by which LAS regulates autophagy in HCC cells, we employed western blotting, Bovine Serum Albumin (BSA) dequenching assay, LysoTracker™ analyses, assessment of autophagic flux, confocal microscopy for LC3 visualization, and transmission electron microscopy for autophagosome visualization. Finally, an HCC xenograft model was established to assess the regulatory effects of LAS on tumor-cell sensitivity to sorafenib in vivo.LAS inhibited the growth and proliferation of HCC cells and induced apoptosis and blocked the G2/M phase in HCC cells. LAS could inhibit autophagy and suppress the generation of autophagic vesicles in HCC cells. It inhibited autophagy initiation by inhibiting the AMPK/mTOR signaling pathway, while simultaneously hindering the fusion of autophagic vesicles with lysosomes by interfering with the mTOR-mediated formation of SNARE complexes, thereby inhibiting autophagy in HCC cells without affecting the number and function of lysosomes. Moreover, it enhanced the sensitivity of HCC cells to sorafenib by inhibiting sorafenib-induced autophagy in vitro and in vivo.We identified an autophagic inhibitor that may serve as a novel therapeutic agent for HCC, potentially opening new avenues in the landscape of HCC treatment.Copyright © 2025 Elsevier GmbH. All rights reserved.