To investigate vitamin D‐related control of brain‐expressed genes, candidate vitamin D responsive elements (VDREs) at ‐7/‐10 kb in human tryptophan hydroxylase (TPH)2 were probed. Both VDREs bound the vitamin D receptor (VDR)‐retinoid X receptor (RXR) complex and drove reporter gene transcription in response to 1,25‐dihydroxyvitamin D₃ (1,25D). Brain TPH2 mRNA, encoding the rate‐limiting enzyme in serotonin synthesis, was induced 2.2‐fold by 10 nM 1,25D in human U87 glioblastoma cells and 47.8‐fold in rat serotonergic RN46A‐B14 cells. 1,25D regulation of leptin (Lep), encoding a serotoninlike satiety factor, was also examined. In mouse adipocytes, 1,25D repressed leptin mRNA levels by at least 84%, whereas 1,25D induced leptin mRNA 15.1‐fold in human glioblastoma cells. Chromatin immunoprecipitation sequencing analysis of the mouse Lep gene in response to 1,25D revealed a cluster of regulatory sites (cis‐regulatory module; CRM) at ‐28 kb that 1,25D‐dependendy docked VDR, RXR, C/EBPp, and RUNX2. This CRM harbored 3 VDREs and single C/EBPβ and RUNX2 sites. Therefore, the expression of human TPH2 and mouse Lep are governed by 1,25D, potentially via respective VDREs located at ‐7/‐10 kb and ‐28 kb. These results imply that vitamin D affects brain serotonin concentrations, which may be relevant to psychiatric disorders, such as autism, and may control leptin levels and affect eating behavior.—Kaneko, I., Sabir, M. S., Dussik, C. M., Whitfield, G. K., Karrys, A., Hseih, J.‐C., Haussler, M. R., Meyer, M. B., Pike, J. W., Jurutka, P. W. 1,25‐dihydroxyvitamin D regulates expression of the tryptophan hydroxylase 2 and leptin genes: implication for behavioral influences of vitamin D. FASEB J. 29, 4023‐4035 (2015). www.fasebj.org