The SPTBN4 gene, a part of the spectrin protein family, plays important roles in various cellular processes, including cell cycle, nerve cell development, and so on. Recently, a new miRNA has been found in this SPTBN4 gene, which was registered at the NCBI database. The aim of the present study was to investigate the expression of this miRNA, called SPTBN4-miR1, in the process of differentiation of human embryonal carcinoma cell line NT2 and also the overexpression effect of this miRNA on the differentiation of these cells. RT-qPCR results indicate that SPTBN4-miR1-5p and SPTBN4-miR1-3p show a significant increase in expression in the process of neural differentiation from day three until the 8^th and 14^th day of differentiation. Then, after overexpressing the SPTBN4-miR1 precursor in NT2 cells and retinoic acid treatment, the expression of pluripotent and differentiation revealed the role of SPTBN4-miR1-5p and SPTBN4-miR1-3p in promoting differentiation and exclusion from the pluripotent state. It seems that by making further studies and finding out the possible targets of these miRNAs, a distinctive marker can be achieved and used to improve the differentiation process.