A novel pathway of vitamin D activation by CYP11A has previously been elucidated. To define the mechanism of action of its major dihydroxy-products, we tested the divergence and overlap between the gene expression profiles of human epidermal keratinocytes treated with either CYP11A1-derived 20,23(OH)2 D3 or classical 1,25(OH)2 D3. Both secosteroids have significant chemical similarity with the only differences being the positions of the hydroxyl groups. mRNA was isolated and examined by microarray analysis using Illumina’s HumanWG-6 chip/arrays and subsequent bioinformatics analyses. Marked differences in the up-and downregulated genes were observed between 1,25(OH)2 D3-and 20,23(OH)2 D3-treated cells. Hierarchical clustering identified both distinct, opposite and common (overlapping) gene expression patterns. CYP24A1 was a common gene strongly activated by both compounds, a finding confirmed by qPCR. Ingenuity pathway analysis identified VDR/RXR signaling as the top canonical pathway induced by 1,25(OH)2 D3. In contrast, the top canonical pathway induced by 20,23(OH)2 D3 was AhR, with VDR/RXR being the second nuclear receptor signaling pathway identified. QPCR analyses validated the former finding by revealing that 20,23(OH)2 D3 stimulated CYP1A1 and CYP1B1 gene expression, effects located downstream of AhR. Similar stimulation was observed with 20(OH)D3, the precursor to 20,23(OH)2 D3, as well as with its downstream metabolite, 17,20,23(OH)3 D3. Using a Human AhR Reporter Assay System we showed marked activation of AhR activity by 20,23(OH)2 D3, with weaker stimulation by 20(OH)D3. Finally, molecular modeling using an AhR LBD model predicted vitamin D3 hydroxyderivatives to be good ligands for this receptor. Thus, our microarray, qPCR, functional studies and molecular modeling indicate that AhR is the major receptor target for 20,23(OH)2 D3, opening an exciting area of investigation on the interaction of different vitamin D3-hydroxyderivatives with AhR and the subsequent downstream activation of signal transduction pathways in a cell-type-dependent manner.