Accumulating evidence suggests that microRNAs and DNA methylation can cause tumor suppressor gene inactivation and promote tumor malignancy. However, the functional mechanisms of miR-29c-3p and DNA methylation in hepatocellular carcinoma (HCC) are unclear. Here, we reported that miR-29c-3p expression was significantly downregulated in HCC tissues and cell lines. Low miR-29c-3p expression correlated with tumor size, multiplicity pathologic features, and shorter overall survival. Overexpression of miR-29c-3p significantly inhibited HCC cell proliferation, apoptosis, migration, and tumor growth in vivo. Moreover, DNA methyltransferases 3B (DNMT3B) was upregulated in HCC tissues, and was negatively correlated with miR-29c-3p expression. Luciferase reporter and western blotting assays revealed that DNMT3B is a target gene directly regulated by miR-29c-3p. Furthermore, miR-29c-3p regulates the methylation of large tumor suppressor gene 1 (LATS1) by DNMT3B, and abnormal methylation of LATS1 inactivates Hippo signaling pathway. We subsequently identified that high DNMT3B expression and low LATS1 expression were frequently identified in HCC tissues and were associated with poor prognosis. In conclusion, our results indicate that miR-29c-3p acts as a tumor suppressor in HCC by targeting DNMT3B and the LATS1-associated Hippo signaling pathway, which might represent a novel potential therapeutic target for HCC.