The control of proliferation and differentiation by tumor suppressor genes suggests that evolution of divergent tumor suppressor repertoires could influence species’ regenerative capacity. To directly test that premise we humanized the zebrafish p53 pathway by introducing regulatory and coding sequences of the human tumor suppressor ARF into the zebrafish genome. ARF was dormant during development, in uninjured adult fins, and during wound healing, but was highly expressed in the blastema during epimorphic fin regeneration after amputation. Regenerative, but not developmental signals resulted in binding of zebrafish E2f to the human ARF promoter and activated conserved ARF-dependent Tp53 functions. The context-dependent activation of ARF did not affect growth and development but prevented regeneration, an unexpected distinct tumor suppressor response to regenerative versus developmental environments. To test the effect of loss of Arf on regeneration, we used Arf deficient mice to investigate digit tip regeneration, a mammalian model of epimorphic regeneration. Regardless of Arf dosage, regeneration was only observed with amputation made in the nail bed. To investigate other tumor suppressors that may influence regeneration, we examined the role of Rb1, a well conserved tumor suppressor, in zebrafish fin regeneration. We identified a differentiation promoting role for the Rb pathway, the pathway surveilled by Arf in mammals, during the redifferentiation phase of epimorphic zebrafish fin regeneration. Finally, in an effort to explore the basis for the divergence of Arf, we performed an evolutionary analysis comparing regenerative capacity, thermal regulation, and Arf expression to discover that ectotherms usually lack Arf and are highly regeneration while endotherms express Arf and not poorly regenerative. We also discovered a role for oxidative stress in zebrafish fin regeneration and ARF activation. Oxidative stress is observed in regeneration but not development and may represent the boost in signaling required for ARF activation during regeneration. These findings highlight the importance of tumor suppressors in regeneration, especially the Rb and p53 pathways. They display the antagonistic pleiotropic characteristics of ARF as both tumor and regeneration suppressor, and they imply that inducing epimorphic regeneration clinically would require modulation of ARF-p53 axis activation.