Chronic hepatitis C virus (HCV) infection is a major cause of hepatocellular carcinoma (HCC) worldwide. The HCV non-structural protein 3 (NS3) protein is considered to affect normal cellular functions and to be involved in HCV carcinogenesis. The expression of microRNA (miRNA) is altered in human HCC, thus implicating its role in hepatocarcinogenesis. To investigate the mechanisms by which the HCV NS3 protein affects the expression of miRNA in malignant hepatocytes, if any, the present study constructed expression vectors encoding the HCV NS3 and NS3/4A proteins, which were stably transfected into HepG2 cells. The biological behaviors of the HepG2 transfectants and their differential expression levels of miRNA expression were investigated. Compared with the HepG2-vector cells, the HepG2-NS3 cells grew at a slower rate, were arrested in the G0/G1 cell cycle phase, formed more colonies and developed larger tumors at a faster rate. Co-expression of HCV NS4A resulted in the inhibition of HCV NS3-stimulated tumorigenicity. A total of 35 miRNAs were dysregulated, 26 of which were downregulated and nine of which were upregulated, in the HepG2-NS3 cells, and 75 miRNAs were altered in HepG2-NS3/4A cells, of which 20 were downregulated and 55 were upregulated). In addition, significant decreases in the mRNA levels of p53 and p21 were observed, which confirmed differential expression of miRNA. These results suggested that differential miRNA profiling in malignant hepatocytes may account for the variable pathophysiological manifestations associated with the HCV NS3 protein. These differentially expressed miRNAs may offer potential as candidates for the development of miRNA-based therapeutics.