Background: <p>Emerging evidence suggests that G-protein-coupled receptor family C group 6 member A (GPRC6A) and Dual oxidase 1 (Duox1) mediate a non-classical testosterone signaling pathway. However, the molecular mechanism in which testosterone mediates the GPRC6A-Duox1 cascade in the hair cycle was unclear. Therefore, this study aimed to elucidate the molecular role of the testosterone-GPRC6A-Duox1 signaling axis in regulating hair cycle progression and testosterone-mediated hair loss.</p> Methods: <p>GPRC6A-deficient and Duox1-deficient keratinocytes were prepared and stimulated with testosterone to assess hydrogen peroxide (H₂O₂) generation. Apoptosis was evaluated in primary keratinocytes from GPRC6A knockout (KO), Duox1 KO, and wild-type (WT) mice. Hair growth cycle progression was examined by measuring anagen phase duration and hair length. Ki-67 expression was analyzed as a marker of the anagen phase. To validate the testosterone-GPRC6A-Duox1 signaling network in androgenetic alopecia, testosterone was topically applied for one week to the back skin of WT, Duox1 KO, and GPRC6A KO mice on postnatal day 31 (P31).</p> Results: <p>GPRC6A-deficient and Duox1-deficient keratinocytes failed to induce H₂O₂ generation in response to testosterone. Testosterone-dependent apoptosis in primary keratinocytes from GPRC6A KO and Duox1 KO mice was suppressed compared to keratinocytes from WT. The anagen phase of the hair growth cycle and hair lengths in GPRC6A KO and Duox1 KO mice were longer than WT consistent with the GPRC6A-Duox1 axis stimulating the anagen-to-catagen transition. The expression of Ki-67, a hallmark of the anagen phase, in GPRC6A KO and Duox1 KO mice was higher than that in WT. Duox1 KO and GPRC6A KO mice were resistant to testosterone-mediated hair loss, unlike WT.</p> Conclusion: <p>Taken together, these results suggest that the GPRC6A-Duox1 axis regulates natural hair cycles and testosterone-mediated hair loss.</p>