The vitamin D receptor (VDR) mediates 1,25-dihydroxyvitamin D3 pleiotropic biological actions through transcription regulation of target genes. The expression levels of this ligand-activated nuclear receptor are regulated by multiple mechanisms both at transcriptional and post-transcriptional levels. Vitamin D3 is the natural VDR activator, but other molecules and signaling pathways have also been reported to regulate VDR expression and activity. In this study, we identify valproic acid (VPA) and natural short-chain fatty acids (SCFAs) as novel transcriptional activators of the human VDR (hVDR) gene. We further report a comprehensive characterization of VPA/SCFA-responsive elements in the 5′ regulatory region of the hVDR gene. Two alternative promoter DNA regions (of 2.4 and 3.8 kb), as well as subsequent deletion fragments, were cloned in pGL4-LUC reporter vector. Transfection of these constructs in HepG2 and human Upcyte hepatocytes followed by reporter assays demonstrated that a region of 107 bp (from −107 to −1) upstream of the transcription start site in exon 1a is responsible for most of the increase in transcriptional activity in response to VPA/SCFAs. This short DNA region is GC-rich, does not contain an apparent TATA box, and includes two bona fide binding sites for the transcription factor Sp1. Our results substantiate the hypothesis that VPA and SCFAs facilitate the activity of Sp1 on novel Sp1 responsive elements in the hVDR gene, thus promoting VDR upregulation and signaling. Elevated hepatic VDR levels have been associated with liver steatosis and, therefore, our results may have clinical relevance in epileptic pediatric patients on VPA therapy. Our results could also be suggestive of VDR upregulation by SCFAs produced by gut microbiota.