Increasing evidences have demonstrated that microRNAs (miRNAs) act an essential role in regulating tumor progression and metastasis. Previous miRNAs microarray data showed that hsa-miR-599 is lower expressed in hepatocellular carcinoma (HCC); however, the function and molecular mechanism of hsa-miR-599 on HCC has not been well illustrated. Here, we first analyzed the expression level of hsa-miR-599 in HCC tissues and cell lines by real-time reverse-transcription PCR (qRT-PCR). Interestingly, we found that hsa-miR-599 was significantly down-regulated in the examined HCC tissues and cell lines. Then cells proliferation, migration and invasion were assessed by MTT, wound-healing and trans-well assay respectively. The results showed that over-expression of hsa-miR-599 resulted in inhibited HCC cells proliferation, migration and invasion in vitro. In addition, dual-luciferase reporter assay, qRT-PCR and Western blot analyzes were used to confirm MYC (v-myc avian myelocytomatosis viral oncogene homolog) as a target gene of hsa-miR-599. MYC expression was up-regulated in HCC tissues and cell lines, and restoration of hsa-miR-599 could remarkably decreased the mRNA and protein levels of MYC. Moreover, over-expression of MYC partly reversed hsa-miR-599-mediated inhibition of HCC cells proliferation, migration and invasion in vitro. Taken together, our data demonstrate that hsa-miR-599 acts as a tumor suppressor and inhibits HCC cells proliferation, migration and invasion by partly targeting oncogenic MYC, which hints that hsa-miR-599 can be a diagnostic and therapeutic biomarker in HCC.