Objective To investigate the mechanism of miR-518a-5p/histone deacetylase 6 (HDAC6) axis in involving in DNA oxidative damage in ovarian cancer (OC) SKOV3 cells. Methods The expression of miR-518a-5p and HDAC6 mRNA in OC tissues and different cancer cells (A2780, SKOV3, CAOV3) was detected by real-time fluorescence quantitative PCR (qRT-PCR). SKOV3 cells were separated into Control group, miR-NC group, miR-518a-5p mimics group, miR-518a-5p mimics+pcDNA-NC group, and miR-518a-5p mimics+pc-HDAC6 group. Cell proliferation and apoptosis were analyzed by plate cloning and Hoechst33258 staining. Immunofluorescence assay was applied to detect the expression of phosphorylated histone H2AX (γ-H2AX). Flow cytometry analysis of reactive oxygen species (ROS) levels. The expressions of HDAC6, Bcl-2-associated X protein (Bax) and B-cell lymphoma-2 (Bcl-2) were analyzed by Western blot. The regulatory relationship between miR-518a-5p and HDAC6 was analyzed by dual luciferase assay. The effect and mechanism of miR-518a-5p on oxidative damage of DNA in OC cells were studied by xenotransplantation tumor model. Results The expression of miR-518a-5p decreased and the expression of HDAC6 increased in OC tissues and A2780, SKOV3 and CAOV3 cells (P<0.001). The expression of miR-518a-5p was the lowest in SKOV3 cells and the expression of HDAC6 was the highest, so SKOV3 cells were selected for follow-up experiments. Compared with the miR-NC group, the miR-518a-5p expression, apoptosis rate, number of γ-H2AX positive cells, relative fluorescence intensity of ROS, and expression of Bax in miR-518a-5p mimics group increased, the expression of HDAC6 mRNA and protein, expression of Bcl-2, and colony formation number decreased (P<0.001). Compared with the miR-518a-5p mimics+pcDNA-NC group, the expression of HDAC6 mRNA and protein, colony formation number, and expression of Bcl-2 in miR-518a-5p mimics+pc-HDAC6 group increased, the apoptosis rate, number of γ-H2AX positive cells, relative fluorescence intensity of ROS, and expression of Bax decreased (P<0.001). HDAC6 had a targeted regulatory relationship with miR-518a-5p. The results of in vivo experiments showed that overexpression of miR-518a-5p decreased tumor volume, weight and HDAC6 protein expression in tumor tissue, and increased positive expression of γ-H2AX (P<0.001). After upregulating HDAC6 expression on the basis of overexpression of miR-518a-5p, graft tumor volume, weight and HDAC6 protein expression were increased, while γ-H2AX positive expression was decreased (P<0.05). Conclusion MiR-518a-5p expression is reduced and HDAC6 expression is increased in OC tissues and cells. Overexpression of miR-518a-5p can induce DNA oxidative damage in SKOV3 cells by inhibiting HDAC6 expression, thereby inhibiting cell proliferation and promoting cell apoptosis.