Recent studies have shown that miR-564 is closely related to the development of various tumors, including breast cancer, lung cancer and glioma. However, few studies have examined miR-564 in hepatocellular carcinoma (HCC). Here, we demonstrated that miR-564 expression in HCC tissues was lower than that in adjacent noncancerous tissues and that miR-564 expression was associated with tumor size, tumor number and vein invasion. Bioinformatics analyses showed that low levels of miR-564 were correlated with poor prognosis. Furthermore, upregulation of miR-564 impaired SMCC7721 and MHCC97H cell proliferation, migration and invasion in vitro and reduced tumorigenesis in vivo. Next, we found that GRB2 was a direct target gene of miR-564 in the HCC cell lines. GRB2 was highly expressed in HCC tissues and negatively correlated with miR-564 expression levels. When GRB2 was downregulated by GRB2-siRNA, HCC cell proliferation, invasion and metastasis were impaired, and restoring GRB2 expression partially reversed the inhibitory effects of miR-564. Western blot analysis showed that miR-564 overexpression reduced GRB2 expression in HCC cell lines and inhibited ERK1/2 and AKT phosphorylation. miR-564 overexpression also upregulated the epithelial-like cell marker E-cadherin and downregulated the interstitial cell-like markers N-cadherin and vimentin. These results suggest that miR-564 inhibits the malignant phenotype of HCC cells by targeting the GRB2-ERK1/2-AKT axis. Consequently, miR-564 may be used as a prognostic marker and therapeutic target for HCC.