Abstract: Objective： To investigate the regulation of miR-509-3p on the proliferation of head and neck squamous cell carcinoma (HNSCC). Methods：Real-time quantitative RT-PCR was used to detected the expression of miR-509-3p in head and neck squamous cell carcinoma cell lines HN4, HN6, HN13, CAL27 and normal oral epithelial cell line HOK. The CAL27 cells were transfected with miR-509-3p mimic and mimic NC, the expression of miR-509-3p was detected by real-time PCR; Cell proliferation experiments including CCK8, colony formation and EDU analysis have been used to verified the effect of miR-509-3p on proliferation. Results：The expression of miR-509-3p was significantly down-regulated in head and neck squamous cell carcinoma cell lines compared with HOK cells; miR-509-3p mimic can significantly increase the expression of miR-509-3p in CAL27. CCK8, colony formation and EDU analysis have shown that overexpression of miR-509-3p can significantly inhibit the proliferation of CAL27 cells. Western blot results showed that the expression levels of EGFR, p-EGFR, PI3K, p-AKT and p-MTOR were significantly decreased in cells transfected with miR-509-3p mimic. Conclusions：MiR-509-3p can affect the proliferation of CAL27 in HNSCC cell CAL27 by regulating the EGFR/PI3K/AKT/MTOR signal pathway.