Regulatory T cells (Tregs) play important immunosuppressive and tissue-regenerative functions in skin. A subset of Tregs localizes to hair follicles (HFs), where they promote hair growth through activation of HF stem cells. However, the mechanisms driving Treg accumulation in HFs remain to be identified. We find that Tregs utilize Cxcr4 to accumulate in HF epithelium and that its expression is partially dependent on glucocorticoid receptor signaling. Additionally, we show that Cxcl12, the main cognate ligand of Cxcr4, is enriched in keratinocytes of the upper HF and that disruption of the Cxcr4-Cxcl12 axis results in suboptimal hair growth. Finally, we demonstrate that upper HF keratinocytes in human skin express Cxcr4 ligands in a pattern similar to that in murine skin. Collectively, these results reveal an evolutionary conserved pathway of Treg trafficking within a barrier tissue that promotes hair regeneration, which may have implications for immunotherapeutic modulation of human alopecia.