Objective: Long noncoding RNAs (lncRNAs) have emerged as new regulators of stem cell pluripotency and neurogenesis. Interestingly, the SOX2 gene, a master regulator of pluripotency and neurogenesis, is embedded within the 3rd intron of an lncRNA, known as SOX2 overlapping transcript (SOX2OT). SOX2DOT (SOX2 distal overlapping transcript) is an isoform of SOX2OT transcribed from a region located >550 kb upstream of SOX2 is expressed in brain Here, we investigated a potential function of SOX2OT in U87-MG (derived from a glioma tumor) and NT2 cells (a human embryonic carcinoma stem cells) which highly expressed SOX2OT and SOX2. Materials and Methods: To explore the hypothesis that SOX2OT has a regulatory role on SOX2 expression, we examined the effect of knocking down SOX2OT in NT2 and U87-MG cells and following that, gene expression and cell cycle alterations were examined. NT2 cells were treated with all-trans retinoic acid (ATRA) during four weeks to induce its differentiation into the neuron-like cells. We then evaluated all ESTs of SOX2OT depositing in GenBank implying for existing of several potential splice variants of SOX2OT. Results: We showed that the lncRNA SOX2OT is a nuclear RNA, and its suppression caused a significant declination of SOX2 expression (0.1, p< 0.01) and also induction of G1 cell cycle arrest and prohibition of S-phase entry in NT2 cells. Using different set of primers we identified several novel splice variants of SOX2OT and SOX2DOT that expressed in NT2 stem cells and U87- MG cells. SOX2OT and SOX2DOT variants revealed controversial expression patterns during the course of differentiation of NT2 cells, suggesting their different potential functional links to the undifferentiated and differentiated state of the NT2 cells respectively. Conclusion: All together, our data suggest a part for SOX2OT and SOX2DOT variants in self-renewal and differetiation of pluripotent stem cells, and a novel regulatory mechanism of SOX2 expression mediated by the lncRNA SOX2OT.