Species such as bony fishes display extensive regenerative capacities, while others such as mammals regenerate poorly. The mechanisms underlying the broad disparity of regenerative capacities in animals remains elusive. Here we report on a comparative epigenomic and transcriptomic approach which identified an evolutionarily conserved regeneration response program (RRP) in vertebrates. By defining the cis-regulomes and transcriptomes of early stages of regeneration in the distantly related zebrafish Danio rerio and the African killifish Nothobranchius furzeri, we were able to discriminate between species-specific and evolutionarily conserved genomic responses to amputation. Importantly, functional testing by systematic transgenic reporter assays of the conserved inhibin beta A (inhba) regeneration responsive enhancer (RRE) from zebrafish, killifish, and humans identified species-specific variations. Furthermore, deletion of the killifish inhba RRE significantly perturbed caudal fin regeneration and completely abrogated cardiac regeneration. We also show that inhba RRE activity requires the presence of predicted binding motifs for the Activator Protein 1 (AP-1) complex. Interestingly, AP-1 binding motifs can be identified in the conserved and non-conserved teleost RREs reported in this study, indicating that AP-1 may be required for both injury and regeneration responses. We propose that changes in RREs driven by natural selection are likely a crucial source of loss of regenerative capacities in vertebrates, including humans.