Objective To investigate the effect of miR6216 on hypoxia/reoxygenation (H/R)induced cardiomyocyte injury. Methods Rat embryonic cardiomyocytes H9C2 were cultured in vitro to construct a H/R (2 h/4 h) model. Realtime quantitative PCR (qRTPCR) was used to detect the expression level of miR6216. Cell counting kit (CCK8) was used to detect cell survival. The plate colony formation assay was used to detect cell clone formation ability. The kit was used to detect intracellular malondialdehyde (MDA) content and activity of lactate dehydrogenase (LDH) in cell culture medium. Apoptosis was detected by flow cytometry. The expression level of cyclin D1 (Cyclin D1), P21, B cell lymphoma/leukemia2 (Bcl2) and Bcl2 related X protein (Bax) were detected by Western blot. The miR6216 inhibitor (antimiR6216) and the miR6216 mimics (miR6216 mimics) were transfected into H9C2, respectively. After the hypoxia/reoxygenation treatment, the above indicators were detected by the above method. Results The expression of miR6216 in H9C2 cells was significantly increased after hypoxia/reoxygenation induction, the cell viability and cloning ability were significantly decreased, MDA content and LDH activity were significantly increased, and the expression of Cyclin D1 and Bcl2 was significantly decreased, and the expression levels of P21 and Bax were significantly increased, and the apoptosis rate was significantly increased (P＜0.05). Inhibition of miR6216 significantly increased H9C2 cell survival and clonality, decreased MDA content and LDH activity, promoted Cyclin D1 and Bcl2 expression, inhibited P21 and Bax expression, and inhibited hypoxiareoxygenationinduced apoptosis (P＜0.05). Overexpression of miR6216 significantly inhibited cell survival and clonality, increased MDA content and LDH activity, inhibited the expression of Cyclin D1 and Bcl2, promoted the expression of P21 and Bax, and aggravated hypoxia/reoxygenation induced apoptosis (P＜0.05). Conclusion miR6216 is upregulated in hypoxia/reoxygenationinduced cardiomyocytes, and inhibition of miR6216 can attenuate hypoxia/reoxygenationinduced cardiomyocyte injury.