MiR‐27 prevents atherosclerosis by inhibiting inflammatory responses induced by lipoprotein lipase. Overexpression of miR‐27b attenuates angiotensin‐induced atrial fibrosis. Nevertheless, studies have rarely investigated on the effect of miR‐27 in cardiomyocyte injury. H9c2 cells were transfected with miR‐27 mimic/inhibitor. Then the cell proliferation was tested by MTT assay and the cell apoptosis was detected by flow cytometry. The luciferase activity assay was utilized to analyze the relationship between miR‐27 and TGFBR1. Quantificational real‐time polymerase chain reaction and western blot were utilized to detect the cardiomyocyte differentiation marker and nuclear factor kappa B (NF‐κB) pathway. Our outcomes demonstrated that miR‐27 expression was downregulated cardiomyocyte injury subjected to hypoxia/reoxygenation (H/R). Additionally, overexpression of miR‐27 could significantly alleviate cardiomyocyte injury by regulating cell activity and apoptosis. The luciferase activity assay confirmed that transforming growth factor ß receptor 1 (TGFBR1) is a direct hallmark of miR‐27. Besides, overexpression of miR‐27 promoted the expression of TGFBR1 in H/R model. After transfection with miR‐27 mimic/inhibitor, the expression of NF‐κB pathway‐related proteins was decreased/increased. Taken together, our data manifested that miR‐27 repressed cardiomyocyte injury induced by H/R via mediating TGFBR1 and inhibiting NF‐κB signaling pathway. Furthermore, miR‐27/ TGFBR1 might be utilized as hopeful biomarkers for myocardial ischemia diagnosis and treatment.