Iroquois homeobox (IRX) gene family, which plays essential roles in embryonic development, has recently been reported to be involved in tumor progression. However, the association of IRX3, a member of the IRX family, with hepatocellular carcinoma (HCC) has not previously been studied. In the present study, we found that IRX3 was upregulated in HCC cell lines (HepG2 and SMMC7721). We investigated the regulatory mechanism of IRX3 in HCC cells. Western blot and luciferase reporter assays identified that IRX3 is a direct target of miR-377. In addition, miR-377 was downregulated in HepG2 and SMMC7721 cell lines, and overexpression of miR-377 inhibited HepG2 cell proliferation, migration and invasion. Moreover, miR-377 restoration significantly abrogated IRX3-induced proliferation, migration and invasion of SMMC7721 cells. These findings demonstrate the tumor-promoting potential of IRX3, and that downregulation of miR-377 may contribute to the upregulation of IRX3 in HCC. The present study provides insights into HCC progression and a novel potential therapeutic target of HCC treatment.