The sodium-dependent multivitamin transporter (SMVT) is essential for mediating and regulating biotin entry into mammalian cells. In cells, holocarboxylase synthetase (HCS) mediates covalent binding of biotin to histones; biotinylation of lysine-12 in histone H4 (K12BioH4) causes gene repression. Here we propose a novel role for HCS in sensing and regulating levels of biotin in eukaryotic cells. We hypothesize that nuclear translocation of HCS increases in response to biotin supplementation; HCS then biotinylates histone H4 at SMVT promoters, silencing biotin transporter genes. We show that nuclear translocation of HCS is a biotin-dependent process that might involve tyrosine kinases, histone deacetylases, and histone methyltransferases in human lymphoid (Jurkat) cells. The nuclear translocation of HCS correlated with biotin concentrations in cell culture media; the relative enrichment of both HCS and K12BioH4 at SMVT promoter 1 (but not promoter 2) increased by 91% in cells cultured in medium containing 10 nmol/L biotin compared with 0.25 nmol/L biotin. This increase of K12BioH4 at the SMVT promoter was inversely linked to SMVT expression. Biotin homeostasis by HCS-dependent chromatin remodeling at the SMVT promoter 1 locus was disrupted in HCS knockdown cells, as evidenced by abnormal chromatin structure (K12BioH4 abundance) and increased SMVT expression. The findings from this study are consistent with the theory that HCS senses biotin, and that biotin regulates its own cellular uptake by participating in HCS-dependent chromatin remodeling events at the SMVT promoter 1 locus in Jurkat cells.