Epigenetic changes, such as alteration of DNA methylation patterns, have been proposed as a molecular mechanism underlying the effect of alcohol on the maintenance of adult stem cells. Epigenetic changes have been proposed as a molecular mechanism underlying the effect of alcohol on the osteogenic potency of adult stem cells. Through genome-wide gene expression microarray and DNA methylome analysis, we identified that lysine specific demethylase 6B (KDM6B), an epigenetic modifier known to be involved in the osteogenic and odontogenic potency in DPSCs, was significantly downregulated by EtOH. We hypothesize that EtOH downregulates the osteogenic potency of DPSCs through KDM6B and its upstream regulatory factors. In order to investigate the upstream and downstream effects of EtOH on the osteogenic potency of DPSCs through KDM6B, we performed a pathway-focused RT-PCR array, western blot, immunofluorescence, and ChIP analyses, and in-vivo experiments. EtOH treatment during osteogenic differentiation of DPSCs suppressed the induction of KDM6B with alterations in the expression of differentiation markers. Knockdown of KDM6B resulted in a marked decrease in mineralization from implanted DPSCs in vivo. Furthermore, an ectopic expression of KDM6B in EtOH-treated DPSCs restored the expression of differentiation-related genes. In addition, we investigated upstream regulators and identified a link between Orai1 and the SMAD1 pathway on KDM6B and osteogenic differentiation. We have identified that osteoinduction decreases Ca2+ influx with a concurrent upregulation in Orai1 and Stim1 expression levels, which are further increased in the presence of EtOH during osteogenic differentiation. We believe the changes observed in Orai1 are indicative of cellular compensation in the presence of osteogenic differentiation and EtOH in order to regulate KDM6B expression and ultimately osteogenic potency. Finally, we have observed that EtOH exposure decreases the osteogenic potential in naï¿½ve DPSCs. Our study has demonstrated that EtOH-induced inhibition of KDM6B plays a role in the downregulation of osteogenic differentiation in DPSCs. This suggests a potential molecular mechanism for cellular insults of heavy alcohol consumption that can lead to decreased mineral deposition potentially associated with abnormalities in dental development and also osteopenia/osteoporosis, hallmark features of fetal alcohol spectrum disorders.