Background: Commonly used to treat inflammatory skin diseases, narrowband ultraviolet B (UVB) has been shown to induce antigen-specific immune suppression when combined with alloantigen immunization, but the underlying mechanism remains elusive. Methods: We used cytometry by time-of-flight (CyTOF) to analyze the peripheral blood mononuclear cells (PBMCs) from 19 psoriasis patients enrolled in UVB trial. Mouse models of ovalbumin (OVA)-induced skin inflammation and allogeneic skin transplantation were used to investigate the effects of UVB on antigen-specific regulatory T cell (Treg) induction. We applied bulk RNA sequencing (RNA-seq) and single-cell RNA sequencing (scRNA-seq) methods to the analysis of mouse skin Tregs and PBMCs, respectively. Results: CyTOF analysis revealed patients’ therapeutic response to be determined by a cluster of CD4 ⁺ T cells expressing T cell receptor (TCR)-activated and Treg-associated molecules. In clinical trial and mouse models of skin inflammation and allogeneic skin transplantation, UVB led to immunosuppressive phenotypes through antigen-specific Treg induction. RNA-seq from mouse skin Tregs showed that UVB enhanced gene expression associated with cell stability, cellular location, and cell proliferation. When compiling with human peripheral Tregs analyzed by scRNA-seq, we found similar gene expression patterns involved in Treg differentiation, maintenance, and function. Furthermore, scRNA-seq analysis also demonstrated that UVB inhibited negative regulators of Treg development, thereby promoting CD4 ⁺ T differentiation into Tregs, clonal expansion of which was also noted. Conclusions: Our findings suggest UVB can induce antigen-specific Tregs in a clinical setting, highlighting its potential for broader immunosuppressive applications.