Primary liver cancer (hepatocellular carcinoma, HCC) is a leading cause of cancer-related deaths, and alternative ways to treat this disease are urgently needed. In recent years, novel approaches to cancer treatment have been based on microRNAs, small non-coding RNA molecules that play a crucial role in cancer progression by regulating gene expression. Overexpression of some microRNAs has shown therapeutic potential, but whether or not this was the case for microRNA-203 (miR-203) in liver cancer was unknown. Therefore, the aim of this study was to investigate the effect of miR-203 overexpression in liver cancer and explore the related mechanisms. Liver cancer cells from the HepG2 and Hep3B cell lines were transfected with either miR-203 mimics or negative control RNA, and then the cells were subjected to cell viability, cell proliferation, and Western blotting assays. As a result of microRNA-203 overexpression, HepG2 and Hep3B cell viability and cell proliferation significantly declined. Furthermore, microRNA-203 overexpression led to inhibited expression of phosphatidylinositol-4,5-bisphosphate 3-kinase (PIK3)/protein kinase B (Akt), c-Jun, and p38 mitogen-activated protein kinases (p38 MAPK), and restored glycogen synthase kinase 3 (GSK 3) activity in HepG2 cells. Our results suggest that c-Jun, p38 MAPK, PIK3CA/Akt, and GSK3 signaling involved in the effect of miR-203 on the proliferation of HCC cells.