Reperfusion following myocardial infarction salvages the ischemic heart but paradoxically exacerbates injury. Yet, efficient treatment for cardiac ischemia/reperfusion injury is still missing in clinics. ATP release through Pannexin1 (PANX1) channels facilitates recruitment of leukocytes to the injured myocardium. Thus, PANX1 channel inhibition might confer cardioprotection. Currently available PANX1 channel blockers lack specificity or in vivo stability. Nanobodies offer a new therapeutic modality given their high target affinity, small size, and deep tissue penetration. Nanobodies targeting Panx1 were recently introduced. Here, their target specificity and selective PANX1 channel inhibition for cardiovascular purposes were validated in vitro. The two most promising candidates were further examined in the context of cardiac ischemia/reperfusion injury. Nanobody-1 (Nb1) and Nb9 reduced neutrophil adhesion to an endothelial monolayer. Nb1 did not affect left ventricular function ex vivo; however, Nb9 tended to diminish the performance of isolated hearts. Finally, in vivo application of Nb1, but not of Nb9 or a control Nb, at the onset of reperfusion increased the survival rate of mice. However, the infarct size observed after treatment with Nb1 was similar than the one found after treatment with the control Nb. In conclusion, Nb1 efficiently and specifically inhibits ATP release from endothelial cells thereby limiting leukocyte adhesion and improving the outcome of cardiac ischemia/reperfusion in mice. This warrants further studies to unveil the detailed molecular mechanism underlying the beneficial effects of Nb1.