Objective: To investigate the effect of miR-551b-3p on apoptosis and radiosensitivity of human lung cancer A549 cells and its molecular mechanism. Methods: miR-NC, miR-551b-3p, anti-miR-NC, anti-miR-551b-3p, si-NC, and si-USP9X were transfected into A549, respectively, and recorded as miR-NC group, miR-551b-3p group, anti-miR-NC group, anti-miR-551b-3p group, si-NC group and si-USP9X group; miR-551b-3p was co-transfected with pcDNA and pcDNA-USP9X into A549, respectively, recorded as miR-551b-3p+pcDNA group and miR-551b-3p+pcDNA-USP9X group. Real-time quantitative PCR (RT-qPCR) was used to detect the expressions of miR-551b-3p and USP9X; Western blotting was used to detect ubiquitin-specific peptidase 9 X-linked (USP9X), B-cell lymphoma/leukemia-2 (Bcl-2), Bcl-2 related X protein (Bax) protein expression; luciferase reporter assay detects the targeting relationship of miR-551b-3p and USP9X; flow cytometry detects apoptosis; cell clone colony formation experiments detect radiosensitivity. Results: The expression of miR-551b-3p was significantly decreased in lung cancer tissues and lung cancer cell A549, and the expressions of USP9X mRNA and protein were significantly increased (all P<0.001). MiR-551b-3p targeted USP9X, overexpressed miR-551b-3p and inhibited USP9X expression, apoptosis rate was significantly increased, Bcl-2 expression was significantly decreased, Bax expression was significantly increased, and cell survival curve was significantly shifted down (all P<0.05). Overexpression of USP9X reversed the effect of miR-551b-3p overexpression on apoptosis and radiosensitivity of lung cancer A549 cells. Conclusion: Overexpression of miR-551b-3p promotes apoptosis of lung cancer A549 cells and enhances radiosensitivity of lung cancer cells. The mechanism may be related to the USP9X.