Background: Remote Ischemic Preconditioning (rIPC) and the antiarrhythmic peptide analogue, Rotigaptide (ZP123), protects against myocardial ischemia-reperfusion injury through potentially similar mechanisms. We aimed to study whether the cardioprotective effects of Rotigaptide and rIPC interacts. Methods: We used male New Zealand White rabbit hearts mounted in a Langendorff system and exposed to 30 min of global no-flow ischemia and 120 min of reperfusion. A total of 48 rabbits were randomized into 6 groups: control (n=6), Rotigaptide (1 μM) before (n=9) or after (n=9) ischemia, rIPC (n=7), rIPC+Rotigaptide before (n=9) or after (n=8) ischemia. rIPC was induced by four cycles of 5-min ischemia and reperfusion on the left hind limb achieved by intermittent tourniquet occlusion. Primary endpoint was infarct size measured by tetrazolium staining. Results: rIPC reduced infarct size compared to controls. Rotigaptide alone did not affect infarct size irrespective of administration before ischemia or during reperfusion. The combination of rIPC and Rotigaptide before ischemia reduced infarct size, whereas the effect of rIPC was abrogated by Rotigaptide when administered during reperfusion. No significant changes in hemodynamic recovery were observed when compared to control group. Conclusion: In contrast to in vivo rIPC, in vitro Rotigaptide did not yield cardioprotection in our rabbit model, but Rotigaptide attenuated the effect of rIPC.