Hepatocellular carcinoma (HCC) is the third-highest cause of cancer-related death in the world. miRNAs have a role in cell division, differentiation, and death biological processes. They are typically dysregulated in cancers, affecting tumor progression. miRNA-296-3p appears to play a crucial role in cancer control, according to new research. However, its expression and roles in HCC are unknown. This study used qRT-PCR and western blotting to detect the miRNA-296-3p and male-specific lethal 2 (MSL2) expression. In addition, cell proliferation, migration, invasion, and apoptosis were studied using CCK-8, flow cytometric analysis, colony formation assay, wound healing test, and transwell assays. The results show that miRNA-296-3p is underexpressed in HCC cell lines, particularly in Huh-7 and HepG2 cells. miRNA-296-3p overexpression lowers the ability of HCC cells to proliferate, migrate, and invade while increasing cell death. Luciferase reporter experiments revealed that the MSL2 is a direct target of miRNA-296-3p. Furthermore, overexpression of miRNA-296-3p reduced MSL2 mRNA and protein levels considerably, according to our findings. Furthermore, the rescue experiments showed that the MSL2 overexpression partially blocked the inhibition effects of miRNA-296-3p mimic on the proliferation and migration of HCC cells. The above results show that miRNA-296-3p may have a repressive effect in HCC by targeting MSL2 and could be used as a therapeutic target for HCC treatment.