Although many cardioprotective interventions have been shown to limit infarct size (IS), in preclinical animal studies of acute myocardial ischemia/reperfusion injury (IRI), their clinical translation to patient benefit has been largely disappointing. A major factor is the lack of rigor and reproducibility in the preclinical studies. To address this, we have established the IMproving Preclinical Assessment of Cardioprotective Therapies (IMPACT) small animal multisite acute myocardial infarction (AMI) network, with centralized randomization and blinded core laboratory IS analysis, and have validated the network using ischemic preconditioning (IPC). Eight sites from the COST Innovators Grant (IG16225) network participated in the IMPACT AMI study. Mice and rats were randomly allocated into Sham, Control, or IPC groups. The IRI group underwent 45 min (mice) or 30 min (rats) of left coronary artery occlusion followed by 24 h reperfusion. IPC comprised three cycles of 5 min occlusion/reperfusion before IRI. IS was determined by a blinded core lab. The majority of site showed significant cardioprotection with IPC. In pooled mouse data, IPC (N = 42) reduced IS/AAR by 35% compared to control (N = 48) (30 ± 16% versus 46 ± 13%; p < 0.005), and in rat data, IPC (N = 36) reduced IS/AAR by 29% when compared to control (N = 39) (32 ± 19% versus 45 ± 14%; p < 0.01). The IMPACT multisite mouse and rat AMI networks, with centralized randomization and blinded core IS analysis, were established to improve the reproducibility of cardioprotective interventions in preclinical studies and to facilitate the translation of these therapies for patient benefit.