Objective To investigate the effect of radiation on cardiomyocyte apoptosis and its related mechanism. Methods Rat H9C2 cardiomyocytes were divided into blank control group, X-ray irradiation group (X-ray group), X-ray irradiation＋microRNA (miRNA)-134-5p inhibitor group (X-inhibitor group) and X-ray irradiation＋miRNA-134-5p inhibitor negative control group (X-NC group). H9C2 cardiomyocytes were irradiated with 6 Gy X-ray, and the changes of various indexes were detected 48 h after irradiation. Cell viability was detected by cell counting kit 8 assay. The apoptosis rate was detected by flow cytometry and Hoechst 33342 staining. The level of reactive oxygen species (ROS) in cells was detected by DCFH-DA fluorescence probe. The mitochondrial membrane potential was detected by JC-1 method. The activity of superoxide dismutase (SOD) and the level of malondialdehyde (MDA) in cells were measured by kits. The expression of miRNA-134-5p was detected by quantitative polymerase chain reaction. The protein expression of brain-derived neurotrophic factor (BDNF), protein kinase B (Akt), phosphorylated Akt (p-Akt), Bcl2 and Bax was detected by Western blotting. Results Compared with the blank control group, in the X-ray group the levels of ROS and MDA were significantly increased, the activity of SOD was significantly decreased, the decreased percentage in mitochondrial membrane potential was significantly increased, the number of micronuclei of DNA damage was significantly increased, and the apoptosis rate was significantly increased (all P＜ 0.01). Compared with the X-ray group, all the indexes of the X-inhibitor group were reversed (P＜0.05 or P＜0.01), while there was no significant difference in the above parameters in the X-NC group (all P＞0.05). Compared with the blank control group, the X-ray group had a significant increase in the miRNA-134-5p level and significant reductions in the protein level of BDNF, Bcl2/Bax ratio, and p-Akt/Akt ratio (all P＜0.01). Compared with the X-ray group, the X-inhibitor group had a significant reduction in the level of miRNA-134-5p and significant increases in the protein level of BDNF, Bcl2/Bax ratio, and p-Akt/Akt ratio (all P＜0.01), and there was no significant difference in all parameters in the X-NC group (all P＞0.05). Conclusion X-ray irradiation can induce oxidative stress, mitochondrial damage, and DNA damage, eventually leading to apoptosis in rat cardiomyocytes, and the mechanism may involve miRNA-134-5p/BDNF/Akt signaling pathway.