The water-soluble, biotin (vitamin B7), is indispensable for normal human health. The vitamin acts as a co-factor for five carboxylases that are critical for fatty acid, glucose and amino acid metabolism. Biotin deficiency is associated with various diseases, and mice deficient in this vitamin display enhanced inflammation. Previous studies have shown that biotin affects the functions of adaptive immune T and NK cells, but its effect(s) on innate immune cells is not known. Because of that and because vitamins such as vitamins A and D have a profound effect on dendritic cell (DC) function, we investigated the effect of biotin levels on the functions of human monocyte derived DCs. Culture of DCs in a biotin deficient medium (BDM) and subsequent activation with LPS resulted in enhanced secretion of pro-inflammatory cytokines, TNF-α, IL-12p40, IL-23 and IL-1β compared to LPS-activated DCs cultured in biotin sufficient (control) and biotin over-supplemented media. Furthermore, LPS-activated DCs cultured in BDM displayed a significantly higher induction of IFN- and IL-17 indicating Th1/Th17 bias in T cells compared to cells maintained in biotin control or over-supplemented media. Investigations into the mechanisms suggested that impaired activation of AMP kinase in DCs cultured in BDM may be responsible for the observed increase in inflammatory responses. In summary, these results demonstrate for the first time that biotin deficiency enhances the inflammatory responses of DCs. This may therefore be one of the mechanism(s) that mediates the observed inflammation that occurs in biotin deficiency.