Background <p>Circular RNAs (circRNAs) are known to contribute significantly to cardiovascular diseases. However, the specific role of circAKT3 in hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury is still unclear.</p> Objective <p>This investigation sought to elucidate the functional significance and underlying molecular pathways of circAKT3 in cardiomyocyte injury triggered by H/R.</p> Methods <p>Human cardiomyocytes (AC16) were treated with H/R. The levels of circAKT3, miR-335-5p, and SP1 were quantified using reverse transcription quantitative polymerase chain reaction (RT-qPCR). To evaluate cellular proliferation and apoptosis, cell counting kit-8 (CCK-8) assays and flow cytometric analysis were employed. Levels of superoxide dismutase (SOD) and reactive oxygen species (ROS), concentrations of inflammatory factors, and markers of myocardial injury creatine kinase myocardial band (CK-MB) and lactate dehydrogenase (LDH) were assayed by enzyme-linked immunosorbent assay (ELISA) and commercial kits. Dual luciferase reporter (DLR) and RNA immunoprecipitation (RIP) are used to confirm targeted relationships between molecules.</p> Results <p>In cardiomyocytes exposed to H/R, circAKT3, and SP1 expression increased significantly, and miR-335-5p expression decreased. CircAKT3 knockdown significantly reduced apoptosis, ROS intensity, CK-MB and LDH release, and tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and IL-6 concentrations. miR-335-5p inhibition partially reversed the anti-apoptotic, ROS-lowering, and enzymatic (CK-MB, LDH) inhibitory effects of circAKT3 knockdown, while concurrently increasing inflammatory cytokine levels. Mechanistically, circAKT3 modulates SP1 expression by competitively binding to miR-335-5p.</p> Conclusion <p>The inhibition of circAKT3 significantly attenuated myocardial injury by downregulating SP1 through targeting miR-335-5p, thereby reducing H/R-induced cardiomyocyte apoptosis, oxidative imbalance, and pro-inflammatory activation.</p>