Extracellular vesicles (EVs) have the potency to function as modulators in the process of myocardial ischemia/reperfusion (I/R) injury. This investigation was performed to decipher the mechanism of human umbilical vascular endothelial cells (HUVECs)-derived EVs in myocardial I/R injury with the involvement of microRNA-129 (miR-129). HUVECs-secreted EVs were collected and identified. An I/R mouse model was developed, and cardiomyocytes were used for vitro oxygen-glucose deprivation/reperfusion model establishment. Differentially expressed miRNAs in myocardial tissues after EV treatment were assessed using microarray analysis. The target relationship between miR-129 and toll-like receptor 4 (TLR4) was identified using a dual-luciferase assay. Gain- and loss-function studies regarding miR-129 were implemented to figure out its roles in myocardial I/R injury. Meanwhile, the activation of the nuclear factor-kappa-binding (NF-κB) p65 signaling and NOD-like receptor 3 (NLRP3) inflammasome was evaluated. EVs diminished the apoptosis of cardiomyocytes and the secretion of inflammatory factors, and all these trends were reversed by miR-129 reduction. miR-129 bound to the 3'-untranslated region of TLR4 directly. The NF-κB p65 signaling and NLRP3 inflammasome were abnormally activated after I/R injury, whose impairment after EVs was partially restored by miR-129 downregulation. This study illustrated that EVs could carry miR-129 to mitigate myocardial I/R injury via downregulating TLR4 and disrupting the NF-κB signaling and NLRP3 inflammasome.