Our recent study of the microRNA (miRNA) expression signature of hypopharyngeal squamous cell carcinoma (HSCC) revealed that microRNA-504 (miR-504) is significantly downregulated in HSCC tissues, suggesting that this miRNA is a candidate tumor suppressor. However, several previous reports indicated that miR-504 has an oncogenic function through targeting TP53. The aim of this study was to investigate the functional significance of miR-504 in cancer cells and to identify novel targets regulated by this miRNA in HSCC cells. First, we confirmed the downregulation of miR-504 in HSCC clinical specimens (P<0.0001) by qPCR. Using two sources of miR-504 to restore function, we observed significant inhibition of cancer cell proliferation in head and neck SCC (HNSCC) cell lines (FaDu, SAS and HSC3) and HCT116 colon carcinoma cells (p53+/+ and p53-/-). In HNSCC cells, induction of cell cycle arrest was observed by miR-504 transfection. To identify the molecular targets of miR-504, we performed gene expression analysis of miR-504 transfectants and in silico database analyses. Our data showed that cell cycle-related genes (RB1, CDK6, CDC23 and CCND1) were candidate target genes of miR-504. In HSCC clinical specimens, the expression of cyclin-dependent kinase 6 (CDK6) was significantly higher in cancer tissues compared to non-cancer tissues (P=0.0004). A significant inverse correlation between CDK6 and miR-504 expression was found (r=-0.43, P=0.0039). Expression of miR-504 inhibited CDK6 expression in HNSCC cells. Loss of tumor-suppressive miR-504 enhanced HSCC cell proliferation through targeting CDK6. The identification of novel tumor-suppressive miR-504-mediated molecular pathways and targets provide new insights into HSCC oncogenesis.