To investigate the effect of silencing myeloid differentiation factor 88 (MyD88) on the activation of NF-κB p65 induced cardiomyocyte apoptosis and inflammatory injury, cells were treated with TNF-α and randomly divided into control group, TNF-α group, TNF-α+shRNA-NC group and TNF-α+sh-MyD88 group. MyD88 protein expression and the phosphorylation of NF-κB p65 in cardiomyocyte were detected by Western blotting; The supernatant levels of creatine kinase MB (CK-MB), myohemoglobin (Mb), cardiac troponin Ⅰ (cTnⅠ), IL-1β, iNOS, IL-6 and IL-10 were detected by ELISA; FACS was used to detect cardiomyocyte apoptosis; Cell proliferation was detected by 5-bromo-2′-deoxyuridine (BrdU) staining; mRNA levels of Ki67, Bcl-2, Bax and c-Myc were detected by RT-PCR. The results showed that compared with the TNF-α+shRNA-NC group, in the TNF-α+sh-MyD88 group, MyD88 protein, CK-MB, Mb and cTnⅠlevels were significantly reduced (all P＜0.05); cardiomyocyte apoptosis rate was significantly decreased (P＜0.05); BrdU positive cell level was significantly increased (P＜0.05); Ki67, c-Myc and the Bcl-2/Bax mRNA levels were significantly increased (all P＜0.05); IL-1β, iNOS, IL-6 levels were significantly decreased (all P＜0.05), IL-10 level was significantly increased (P＜0.05); and p-p65/p65 ratio was significantly decreased (P＜0.05). These results suggest that silencing MyD88 has a protective effect on TNF-α-induced cardiomyocyte injury via inhibiting NF-κB p65 activation.