The new claim about the origin of cancer known as Cancer Stem Cell theory states that a somatic differentiated cell can dedifferentiated or reprogrammed for regaining the cancer cell features. It has been recently shown that expression of stemness factors such as Oct4, Sox2, Nanog and Klf4, in a variety of somatic cancers can leads to development of tumorogenesis. Here, the expression of Oct4 variants were evaluated in brain tumor tissues by quantitative RT-PCR and immunohistochemical (IHC) analysis. In next phase of our study, the expression of Oct4B1 was knock-down in brain cancer cell lines and its effect on cell cycle was assessed. Finally, in order to get insights into sequence-structure-function relationships of Oct4 isofroms, their sequences were analysed using bioinformatic tools. Our data revealed that all three variants of Oct4 are expressed in different types of brain cancer. The expression level of Oct4B1, in contast to Oct4B, was much higher in high-grade brain tumors compared with low-grade ones. In line with qPCR, the expression of Oct4A and B isofroms was confirmed with IHC in different types of brain tumors. Moreover, as a result of the suppression of Oct4B1 expression, the brain cancer cells were arrested in G2/M phase of cell cycle. Bioinfromatics data indicated that the predicted Oct4B1 protein have DNA binding properties. All together, our findings suggest that Oct4B1 has a potential role in tumorigenesis of brain cancer and can be considered as a new tumor marker with potential value in diagnosis and treatment of brain cancer.