Background Cardiac microvascular endothelial cells (CMECs) are rapidly damaged after myocardial ischemia or hypoxia. In this study, we intend to explore whether ursolic acid (UA) can protect CMECs against hypoxia/reoxygenation (H/R) injury and to detect related molecular mechanism. Methods CMECs were subjected to H/R condition in the absence or presence of UA. Cell behaviors were measured by Cell Counting Kit-8, transwell, ELISA and western blot assays. siRNA was applied to reduce ICAM1 expression, then the effect of co-treatment of UA and si-ICAM1 on CMECs has been detected by biological experiments. Results Under H/R stimulation, the proliferation and migration of CMECs were inhibited, as well as the inflammation and oxidative stress were enhanced. UA treatment obviously reversed these H/R-induced injuries and reduced ICAM1 expression. Moreover, knockdown of ICAM1 could alleviate the H/R-induced injuries and strengthen the protective effect of UA on CMECs under H/R condition. Additionally, the protein levels of TLR4, MyD88 and p-P65 NF-κB were obviously increased after H/R stimulation, whereas the addition of UA could alter the phenomena by reducing TLR4, MyD88, and p-P65 NF-κB expression. Conclusions Our results insinuated that UA could alleviate H/R-induced injuries in CMECs by regulating ICAM1 and TLR4/MyD88/NF-κB pathway.