Previously, we reported that ultraviolet B-radiation (UVR) suppressed Bacillus Calmette–Guérin (BCG) vaccine-induced resistance to Mycobacterium tuberculosis in guinea pigs (GP). Herein, we investigated the cellular and molecular changes within the irradiated GP epidermis and the in vivo effect of supernatants from UV-irradiated (200 J m−2) epidermal cells (UV-sup) on M. bovis BCG vaccination. UVR increased the number of nucleated keratinocytes in the skin, but caused a decrease in the proportions of CD25+T cells. In the spleen, UVR resulted in a decrease in the proportions of T-cell subsets including CD25+T cells, and major histocompatibility complex (MHC) class II+ and CD14+ cells. Similarly, significant up-regulation of several cytokine mRNAs including IL-10 was also observed. Furthermore, UV-sup significantly reduced the MHC class II expression in peritoneal cells and reduced T-cell proliferation to ConA. The proliferation to purified protein derivative (PPD) was restored to normal levels by anti-IL-10 antibody. The UV-sup when injected into BCG-vaccinated GP significantly diminished the skin test response and T-cell proliferation to PPD and up-regulated the expression of IL-10, IL-4, IL-1β and Foxp3 mRNAs in the lymph node or spleen. Thus, whole body UVR induces profound cellular and molecular changes and injection of UV-sup from epidermal cells mimics the effect of whole body UVR in BCG-vaccinated GP.