© 2017 The Author(s). Using LC/qTOF-MS we detected lumisterol, 20-hydroxylumisterol, 22-hydroxylumisterol, 24-hydroxylumisterol, 20,22-dihydroxylumisterol, pregnalumisterol, 17-hydroxypregnalumisterol and 17,20-dihydroxypregnalumisterol in human serum and epidermis, and the porcine adrenal gland. The hydroxylumisterols inhibited proliferation of human skin cells in a cell type-dependent fashion with predominant effects on epidermal keratinocytes. They also inhibited melanoma proliferation in both monolayer and soft agar. 20-Hydroxylumisterol stimulated the expression of several genes, including those associated with keratinocyte differentiation and antioxidative responses, while inhibiting the expression of others including RORA and RORC. Molecular modeling and studies on VDRE-transcriptional activity excludes action through the genomic site of the VDR. However, their favorable interactions with the A-pocket in conjunction with VDR translocation studies suggest they may act on this non-genomic VDR site. Inhibition of RORα and RORγ transactivation activities in a Tet-on CHO cell reporter system, RORα co-activator assays and inhibition of (RORE)-LUC reporter activity in skin cells, in conjunction with molecular modeling, identified RORα and RORγ as excellent receptor candidates for the hydroxylumisterols. Thus, we have discovered a new biologically relevant, lumisterogenic pathway, the metabolites of which display biological activity. This opens a new area of endocrine research on the effects of the hydroxylumisterols on different pathways in different cells and the mechanisms involved.