: In contrast to cardiomyocytes, autophagy in cardiac microvascular endothelial cells (CMECs) during ischemia/reperfusion (I/R) injury has not been fully investigated. Tongxinluo (TXL), a traditional Chinese medicine, was shown to be vascular protective. We aimed to elucidate the role of autophagy and its regulatory mechanisms by TXL in CMECs subjected to I/R injury. CMECs were exposed to different treatments for 30 minutes and subjected to hypoxia/reoxygenation each for 2 hours. The results indicated that hypoxia/reoxygenation significantly induced autophagy, as identified by an increased number of monodansylcadaverine-positive CMECs, increased autophagosome formation, and a higher type II/type I of light chain 3 ratio, but not Beclin-1 expression. Autophagy inhibition using 3-methyladenine was proapoptotic, but rapamycin-induced autophagy was antiapoptotic. TXL enhanced autophagy and decreased apoptosis in a dose-dependent manner, reaching its largest effect at 800 μg/mL. 3-methyladenine attenuated the TXL-promoted autophagy and antiapoptotic effects, whereas rapamycin had no additional effects compared with TXL alone. TXL upregulated mitogen-activated protein kinase and extracellular signal-regulated kinase (ERK) phosphorylation; however, PD98059 abrogated ERK phosphorylation and decreased autophagy and increased apoptosis compared with TXL alone. These results suggest that autophagy is a protective mechanism in CMECs subjected to I/R injury and that TXL can promote autophagy through activation of the mitogen-activated protein kinase/ERK pathway.