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Objective: To investigate the regulatory mechanism of miR-216a-5p on autophagy and radiosensitivity of gastric cancer cell and its regulatory effect on dual-specificity phosphatase 10 (DUSP10). Methods: SGC-7901 cells were irradiated with linear accelerator 6-MV X-ray at a dose rate of 0.8 Gy/min. The total dose is 8Gy. miR-216a-5p-mimic, NC-mimic, pcDNA-DUSP10 or pcDNA-NC were transfected into SGC-7901 cells with Lipofectamine 2000 reagent. After transfection, the cells were divided into miR-216a-5p-mimic group and NC-mimic group, and each group was divided into two subgroups, 0Gy and 8Gy. In the rescue experiment, the cells were divided into miR-216a-5p mimic+pcDNA DUSP10 group and miR-216a-5p mimic+pcDNA NC group. The mRNA levels of miR-216a-5p and DUSP10 were detected by qRT-PCR. Cell proliferation was detected by 5-ethynyl-2'-deoxyuridine (EdU) incorporation test and colony formation assay. The apoptosis was assessed by flow cytometry. The protein expressions of DUSP10, Bax, Bad, Bcl-2, LC3 and p62 were detected by Western blot. The expression of γH2AX was detected by immunofluorescence method to evaluate DNA double-strand break (DSB) in cells. The targeting relationship between miR-216a-5p and DUSP10 was detected by luciferase reporter gene. Autophagosomes were detected by GFP-mRFP-LC3. Results: Compared with the 8Gy NC-mimic group, the number of colonies, EdU positive rate and Bcl-2 protein expression level in the 8Gy miR-216a-5p-mimic group decreased, while the positive rate of γH2AX, the rate of apoptosis and the expression levels of Bax and Bad proteins increased (P<0.01). Compared with the 8Gy NC-mimic group, the number of autophagosomes and the expression level of LC3II protein in the 8Gy miR-216a-5p-mimic group decreased, while the expression level of p62 protein increased (P<0.001). After co-culture with miR-216a-5p-mimic, the relative luciferase activity of DUSP10-3'-UTR-WT was significantly reduced compared with the DUSP10-3'-UTR-MUT group (P<0.001). Compared with the NC-mimic group, the DUSP10 mRNA and protein expression levels in the miR-216a-5p-mimic group were lower (P<0.001). Compared with the miR-216a-5p mimic+pcDNA NC group, the number of colonies and autophagosomes in the miR-216a-5p mimic+pcDNA DUSP10 group increased, while the apoptosis rate was decreased (P<0.001). Conclusion: miR-216a-5p inhibits cell proliferation, increases radiation-induced apoptosis and inhibits radiation-induced autophagy by inhibiting DUSP10, thereby enhancing the radiosensitivity of gastric cancer cells. [ABSTRACT FROM AUTHOR]