Objective To investigate the targeting relationship of miR-149-5p and fibroblast growth factor 21 (FGF21), and the effect of miR-149-5p on hypoxia/reoxygenation (H/R) injury of rat`s cardiomyocytes. Methods Ten male C56BL/6J mice were used to construct the cardiac ischemia-reperfusion (I/R) models, and qRT-PCR was performed to detect the changes of miR-149-5p levels in the heart tissues of I/R mice. H9c2 cells cultured to logarithmic phase were divided into control group, H/R group, H/R+miR-149-5p inhibitor group, H/R+miR-149-5p NC group, H/R+miR-149-5p mimics group and H/R+miR-149-5p mimics+FGF21 group, and induced H/R model and transfection treatment. The activities of lactate dehydrogenase (LDH) and creatine kinase (CK) in H9c2 cardiomyocytes were detected by ELISA; Cell viability was detected by MTT assay; apoptosis was detected by flow cytometry; the level of miR-149-5p was detected by qRT-PCR; the expression levels of Bcl-2, Bax, cleaved caspase-3 and FGF21 proteins were detected by Western blotting; the targeting relationship between miR-149-5p and FGF21 were determined by dual luciferase assay. Results Compared with the sham operation group, the level of miR-149-5p in the heart tissue of I/R mice was significantly increased (P<0.05). Compared with those in control group, the apoptosis rate, LDH and CK activities and Bax, cleaved caspase-3 protein expression levels of H9c2 cells in H/R group were significantly increased (P<0.05), cell viability and FGF21, Bcl-2 protein expression levels decreased significantly (P<0.05). Compared with those in H/R group and H/R+miR-149-5p NC group, the apoptosis rate, LDH and CK activities and Bax, cleaved caspase-3 protein expression levels of H9c2 cells in H/R+miR-149-5p inhibitor group were significantly decreased (P<0.05), cell viability and FGF21, Bcl-2 protein expression levels were significantly increased (P<0.05). Compared with those in H/R group, the apoptosis rate, LDH and CK activities and Bax, cleaved caspase-3 protein expression levels of H9c2 cells in H/R+miR-149-5p mimics group were significantly increased (P<0.05), cell viability and FGF21, Bcl-2 protein expression levels decreased significantly (P<0.05). Compared with those in H/R+miR-149-5p mimics group, the apoptosis rate, and LDH and CK activities of H9c2 cells in miR-149-5p mimics+FGF21 group were significantly decreased (P<0.05), cell viability was significantly increased (P<0.05). The result of dual luciferase assay indicated the targeted regulatory relationship between miR-149-5p and FGF21. Conclusions MiR-149-5p is up-regulated significantly in the myocardial tissue of I/R mice, and negatively regulate the level of FGF21, leads to decreased cell viability and increased apoptosis of H/R-treated rat's cardiomyocytes.