Myocardial ischemia-reperfusion injury (MIRI) exacerbates myocardial damage, resulting in a further decline in heart function. Abnormally high TRAF6 expression was reported in MIRI. However, what actually causes the abnormally elevated expression of TRAF6 in MIRI is still unclear and needs further study.Mice underwent left anterior descending (LAD) occlusion for 30 minutes and then reperfusion for 24 hours to establish our MIRI model. Hypoxia/reoxygenation (H/R) was used to treat AC16 cells. Morphology of myocardial tissues was examined by HE staining and myocardial infarction size was measured using Evans blue/TTC staining. The levels of vital molecules were determined using RT-qPCR, ELISA, and western blot. The interactions between molecules were validated by Co-IP. Cell viability and apoptosis were evaluated using CCK-8 and flow cytometry.Our results showed higher expressions of TRAF6 and SUMO1 in MIRI models. In addition, TRAF6 protein was modified by SUMOylation, thereby enhancing TRAF6 protein stability and TRAF6 expression. Of note, TIF1β mediated TRAF6 protein SUMOylation. Moreover, TRAF6 overexpression reversed TIF1β silencing-generated enhancement of cell viability and reductions in cell apoptosis and inflammatory response in H/R-induced AC16 cells.TIF1β mediated TRAF6 protein SUMOylation to stabilize TRAF6 protein and enhance TRAF6 level, thereby facilitating MIRI through inhibiting cell viability and enhancing cell apoptosis and inflammatory response.