For patients with head and neck squamous cell carcinoma (HNSCC), survival rates have not improved due to local recurrence and distant metastasis. Current targeted molecular therapies do not substantially benefit HNSCC patients. Therefore, it is necessary to use advanced genomic approaches to elucidate the molecular mechanisms underlying the aggressiveness of HNSCC cells. Analysis of our microRNA (miRNA) expression signature by RNA sequencing showed that the miR-199 family (miR-199a-5p, miR-199a-3p, miR-199b-5p and miR-199b-3p) was significantly reduced in cancer tissues. Ectopic expression of mature miRNA demonstrated that all members of the miR-199 family inhibited cancer cell migration and invasion by HNSCC cell lines (SAS and HSC3). These findings suggested that both passenger strands and guide strands of miRNA are involved in cancer pathogenesis. In silico database and genome-wide gene expression analyses revealed that the gene coding for integrin α3 (ITGA3) was regulated by all members of the miR-199 family in HNSCC cells. Knockdown of ITGA3 significantly inhibited cancer cell migration and invasion by HNSCC cells. Moreover, overexpression of ITGA3 was confirmed in HNSCC specimens, and high expression of ITGA3 predicted poorer survival of the patients (P = 0.0048). Our data revealed that both strands of pre-miR-199a (miR-199a-5p and miR-199a-3p) and pre-miR-199b (miR-199b-5p and miR-199b-3p) acted as anti-tumor miRNA in HNSCC cells. Importantly, the involvement of passenger strand miRNA in the regulation of cellular processes is a novel concept in RNA research. Novel miRNA-based approaches for HNSCC can be used to identify potential targets for the development of new therapeutic strategies.