microRNAs (miRNAs) are known to be involved in the pathogenesis of hepatocellular carcinoma (HCC). miR-128-3p was recently reported to be deregulated in several types of cancer. However, the biological function and potential mechanisms of miR-128-3p in HCC remain unknown. In the present study, we found that miR-128-3p was frequently downregulated in HCC tissues and cell lines by qRT-PCR analysis. Moreover, functional assays showed that overexpression of miR-128-3p markedly suppressed HCC cell proliferation by inducing G1 phase cell arrest and migration. Mechanistically, miR-128-3p was confirmed to regulate PIK3R1 (p85α) expression thereby suppressing phosphatidylinositol 3-kinase (PI3K)/AKT pathway activation using qRT-PCR and western blot analysis. Furthermore, correlation analysis and Kaplan-Meier estimates revealed an inverse correlation between miR-128-3p and p85α as well as a shorter disease-free survival (DFS) period after HCC resection in patients with low miR-128-3p expression. Hence, we conclude that miR-128-3p, which is frequently downregulated in HCC, inhibits HCC progression by regulating PIK3R1 and PI3K/AKT activation, and is a prognostic marker for HCC patients.