Integrin αvβ6 is an epithelial-specific receptor that binds and activates latent transforming growth factor-β1 (TGF-β1). TGF-β1 has been implicated as an endogenous inducer of hair follicle (HF) regression during hair cycling. We hypothesized that αvβ6 integrin-mediated TGF-β1 signaling regulates hair regeneration and HF involution. In wild-type (WT) mice, the expression of integrin αvβ6 was strongly upregulated in the outer root sheath (ORS) during early hair regeneration, and was specifically enhanced in the HF bulge region. Expression gradually decreased in late anagen and remained restricted to the bulge region in the catagen and telogen stage HFs. The first spontaneous hair cycle was not altered in β6 integrin knockout (β6(-/-)) mice. However, after depilation, β6(-/-) mice exhibited retarded HF regression compared with WT controls. β6(-/-) follicles contained significantly higher numbers of proliferating Ki67-positive keratinocytes than WT follicles at an identical cycle stage. The β6(-/-) follicles also demonstrated significantly reduced levels of TGF-β1 expression and Smad2 phosphorylation during early anagen and anagen-catagen transition. Our study indicates that αvβ6 integrin has an important inhibitory role in keratinocyte proliferation in both HFs and interfollicular epidermis. Thus, downregulated TGF-β1 signaling in β6(-/-) mice may affect bulge niche stem cell behavior.