Sorafenib is currently the only systemic agent approved for treatment of advanced hepatocellular carcinoma (HCC). However, intrinsic and acquired resistance to sorafenib remains a great challenge with respect to improving the prognoses of patients with HCC. The cyto-protective functions of autophagy have been suggested as a potential mechanism by which chemoresistance or targeted drug resistance occurs in tumour cells. In the present study, miR-142-3p was identified as a novel autophagy-regulating microRNA (miRNA) that plays a vital role in sorafenib resistance in HCC cells. Gain- and loss-of-function assays revealed that ectopic miR-142-3p upregulation sensitized HCC cells to sorafenib by reducing sorafenib-induced autophagy, enhancing sorafenib-induced apoptosis and inhibiting cell growth, whereas miR-142-3p inhibition exerted contrasting effects. Bioinformatics analysis and luciferase reporter and rescue assays showed that autophagy-related 5 (ATG5) and autophagy-related 16-like 1 (ATG16L1) are potential targets through which miR-142-3p regulates autophagy inhibition. Furthermore, we verified that PU.1 regulated the expression of miR-142-3p in conjunction with our cellular experiments and the related results in the literature. Our findings show that targeting the PU.1-miR-142-3p-ATG5/ATG16L1 axis may be a useful therapeutic strategy for preventing cyto-protective autophagy to overcome sorafenib resistance.