OBJECTIVE: To investigate the impact of cyclic RNA (circRNA) ATPase phospholipid transporting 9B gene (circATP9B) on hypoxia/reoxygenation (H/R)-induced cardiomyocyte (H9C2) injury and the possible mechanism. STUDY DESIGN: RT-qPCR was adopted for quantification of circATP9B and miR-525-5p in H/R-induced H9C2 cells, and dual luciferase reporter and RT-qPCR assays were used for targeting relationship verification between the two. H9C2 cells were assigned to the control, H/R, H/R+si-NC, H/R+si-circATP9B, H/R+ miR-NC, H/R+miR-525-5p, H/R+si-circATP9B+antimiR-NC, and H/R+si-circATP9B+anti-miR-525-5p groups. Then flow cytometry was utilized for cell apoptosis determination, and corresponding kits were used for quantification of MDA content and SOD and GSH-Px activities. RESULTS: CircATP9B bound to miR-525-5p directly and negatively regulated miR-525-5p. In contrast to the control group, the H/R group presented notable increases in circATP9B expression, apoptosis rate, and MDA content and notable decreases in miR-525-5p expression, and SOD and GSH-Px activities. The H/R+ si-circATP9B group presented greatly lower MDA concentration and apoptosis and greatly higher GSH-Px and SOD activities than the H/R+si-NC group. Additionally, the H/R+miR-525-5p group showed greatly lower MDA concentration and apoptosis and greatly higher GSH-Px and SOD activities than the H/R+miR-NC group, and the H/R+si-circATP9B+anti-miR-525-5p group presented notably higher MDA concentration and apoptosis and greatly lower GSH-Px and SOD activities than the H/R+si-circATP9B+anti-miR-NC group. CONCLUSION: Interfering with circATP9B can alleviate apoptosis as well as oxidative stress injury of H/R-induced cardiomyocytes via negative regulation on miR-525-5p. © Science Printers and Publishers, Inc.