MiR-381-3p and nuclear autoantigenic sperm protein (NASP) have regulatory functions in tumors. Whether NASP is targeted by miR-381-3p to influence biological characteristics of cancer in head-neck squamous cell carcinoma (HNSCC) cells was investigated. StarBase (version 3.0) found that the expression of NASP was increased with the down-regulation of miR-381-3p in laryngocarcinoma tissue, AMC-HN-3，FaDu，HNE-3，and Detroit 562 cell lines. MiR-381-3p could target NASP, reduce the expression of MMP-2 and MMP-9, Vimentin, repress the cell viability, invasion, and migration, and promote the expression of E-cadherin in AMC-HN-3 cells. Overexpressed NASP could increase the viability, migration and invasion rates in AMC-HN-3 cells, which could be partially reversed by overexpressed miR-381-3p. Thus, miR-381-3p targeted and suppressed NASP gene, reduced the viability, migration, invasion, EMT of HNSCC cells, demonstrating that miR-381-3p has the potential to be a therapeutic target in inhibiting the progression of HNSCC. Overexpression of miR-381-3p reversed the promotive effect of NASP on the viability, migration, and invasion of AMC-HN-3 cells.