Sepsis often leads to systemic multiple organ dysfunction, with the majority of deaths attributable to acute myocardial injury (AMI). In this study, we aimed to explore the functional role of miR-365a-3p in sepsis-induced AMI. The sepsis myocardial injury model was constructed using lipopolysaccharide (LPS) both in vitro and in vivo with selective regulation of miR-365a-3p expression. Real-time PCR or Western blot was employed to detect the expressions of miR-365a-3p, inflammatory cytokines (tumor necrosis factor alpha (INF alpha), interleulcin-1 beta (IL-1 beta), and IL-6), and inflammation-related proteins (nudear factor-kappa B (NF-kappa B), I-kappa B, myeloid differentiation factor 88 (MyD88)) in myocardial tissues and cells. Also, cell counting kit-8 (CCK8) and flow cytomeny assays were used to measure cardiomyocyte proliferation and apoptosis, respectively. Furthermore, the targeting relationship between miR-365a-3p and MyD88 was verified with the dual luciferase activity assay. miR-365a-3p was downregulated in LPS-induced myocardial injury model. miR-365a-3p overexpression attenuated cardiomyocyte apoptosis and suppressed the expressions of inflammatory cytokines and proteins. Inhibiting miR-365a-3p, however, produced the opposite effects. Mechanistically, miR-365a-3p targeted the 3'-untranslated region of MyD88, thereby inactivating MyD88-mediated NF-kappa B pathway. miR-365a-3p overexpression mitigated sepsismediated myocardial injury by inhibiting MyD88-mediated NIF-kappa B activation.