Background: Hepatocellular carcinoma (HCC) is one of the most prevalent malignant tumors in clinical practice, with hepatitis B virus (HBV) being the most common risk factor for HCC. MicroRNAs (miRNAs) have emerged as a new marker for disease diagnosis and molecularly targeted therapies; however, the mechanism of miR 642a-5p in HBV-associated HCC remains unclear. Objectives: The aim of this study was to investigate the expression of miR 642a-5p, which targets DNA damage-inducible transcript 4 (DDIT4), in HBV-associated HCC, and its effect on the proliferation, migration, and invasion of HBV-positive HCC cells. Methods: miR 642a-5p in the serum of patients with HBV-associated liver cancer (LC), as well as miR 642a-5p and DDIT4 mRNA in LC tissues and cells, and HBV DNA in HBV-positive cells were detected. The targeting of DDIT4 by miR 642a-5p and the progression of cells were also examined. All cell experiments were repeated five times. Results: The results indicated that levels of miR 642a-5p were decreased, while levels of DDIT4 were increased in the serum, tissues, and cells of HBV-positive HCC patients. Overexpression of miR 642a-5p inhibited the progression of HBV-positive HCC cells, suppressed HBV DNA replication, cell proliferation, and invasion, and promoted apoptosis in HepG2.2.15 cells. Conclusions: In addition, miR 642a-5p directly targeted DDIT4, and knockdown of DDIT4 reversed the effects of miR 642a-5p upregulation, promoting the progression of HBV-positive HCC cells. In conclusion, miR 642a-5p is expressed at low levels in HBV-associated HCC and inhibits HBV DNA replication and tumor progression in HBV-positive HCC by targeting DDIT4. This study provides a foundation for molecular targeted therapy in HBV-positive HCC.