Cardiomyocytes, macrophages, and fibroblasts play important roles in inflammation and repair during myocardial ischemia/reper fusion injury (MIRI). Myeloid differentiation primary response 88 (MyD88) is upregulated in immunocytes, cardiomyocytes, and fibroblasts during MIRI. MyD88 induces the secretion of proinflammatory cytokines, including interleukin (IL)-1 beta, IL-6, and tumor necrosis factor alpha (TNF-alpha), while fibroblasts are recruited and activated to mediate cardiac remodeling. The aim of this study was to assess the anti-MIRI effect and mode of action of the novel MyD88 inhibitor TJ-M2010-5. We synthesized TJ-M2010-5 and identified its target by co-immunoprecipitation, after which we established a murine MIRI model and tested the protective effect of TJ-M2010-5 by immunohistochemistry, flow cytometry, real-time PCR, and western blotting. Neonatal rat cardiomyocytes subjected to anoxia/reoxygenation were also isolated and their supernatants used to stimulate cardiac macrophagocytes and fibroblasts in vitro. MyD88 was found upregulated during the early and late phases after MIRI. The MyD88 inhibitor considerably improved cardiac function, reduced cardiomyocyte apoptosis, reduced IL-1 beta, IL-6, and TNF-alpha secretion, and inhibited CD80+CD86+MHCII+ macrophage and fibroblast migration. Moreover, TJ-M2010-5 markedly inhibited Toll-like receptor/MyD88 signaling in vivo and in vitro. Thus, our findings highlight TJ-M2010-5 as a potential therapeutic agent for MIRI treatment.