The effects of ATP receptor agonists/antagonists on skin barrier recovery rate were evaluated in hairless mice. Topical application of ATP and α,β-methylene ATP (agonist of P2X receptor) delayed barrier recovery. Topical application of suramin (nonspecific ATP receptor antagonist), pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) (P2X receptor antagonist), and 2′,3′-O-(2,4,6-trinitrophenyl)adenosine 5′-triphosphate (TNP-ATP) (P2X1, P2X3, P2X2/3 antagonist) after barrier disruption accelerated the barrier repair. The P2Y type receptor antagonist Reactive Blue 2 did not affect the barrier repair process. Moreover, topical application of TNP-ATP prevented epidermal hyperplasia induced by barrier insult under low environmental humidity. ATP was secreted immediately after tape stripping on skin in organ culture. α,β-Methylene ATP increased intercellular calcium in cultured keratinocytes and the increase was blocked by TNP-ATP. Both reverse transcription polymerase chain reaction assay and immunohistochemical study showed the existence of protein that had a structure similar to P2X3 on hairless mouse epidermis. These results suggest that cutaneous barrier homeostasis can be regulated by cation flux through a P2X3-like ATP receptor.