Our previous work identified downregulated miR-483-5p in hepatocellular carcinoma (HCC). This study aims to identify the target of miR-483-5p, evaluate the potential value of this target as a tumor marker for HCC and explore the role of this target in HCC tumorigenesis. Upregulated retinoic acid induced 16 (RAI16) (17/18 cases) was negatively correlated with downregulated miR-483-5p (14/18 cases) in HCC tissues. The dual-luciferase reporter assay showed that RAI16 is a target of miR-483-5p. Immunohistochemistry analysis showed RAI16 was moderate or strong staining in tumor tissues but negative or weak staining in adjacent non-tumor tissues. The sensitivity and specificity of RAI16 for HCC diagnosis were 70.6 and 93.6%, respectively, and increased to 80.9 and 92.0% when combined with glypican-3. Finally, overexpression or knockdown of RAI16 increased or decreased cell viability and colony formation in HCC cell lines and enhanced or inhibited tumor cell growth in xenograft nude mice. Mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) and transforming growth factor-β pathways were mostly affected by RAI16. RAI16 could activate the phosphorylation of ERK1/2 and SMAD2/3. In conclusion, RAI16 may serve as a useful therapeutic agent for HCC gene therapy and tumor marker for HCC diagnosis.