【Abstract】Objective To investigate the regulation of proliferation and differentiation of brain glioma stem cells (BGSCs) by HOTAIR via STAT3/NANOG signaling pathway. Methods U351 stem cells were transferred into siHOTAIR and Controlsi sequences, respectively. The blank control group was set up. HOTAIR gene expression, cell proliferation and cell cycle distribution were detected by RTqPCR, MTT and flow cytometry. After induction of differentiation, the percentage of CD133, glial acid fibrin (GFAP), βmicrovascular protein Ⅲ(βTublin Ⅲ) and myelin basic protein (MBP) positive cells, the expressions of leukemia inhibitory factor (LIF), STAT3, NANOG mRNA and protein and pSTAT3 protein were detected by RTq PCR and Western blot. Results Compared with blank control group and noload group, the relative expression of HOTAIR gene downregulated, the OD value of MTT experiment decreased, the G0/G1 phase of cell cycle decreased and the S, G2/M phase increased, the percentage of CD133, GFAP, βTublin Ⅲ and MBP positive cells increased, while the relative expressions of LIF, NANOG mRNA and protein and pSTAT3/STAT3 decreased in the interference group (P＜005). There were no significant differences in the above indexes between the groups (P＞005). Conclusion Silencing HOTAIR gene can effectively inhibit the proliferation and promote the differentiation of BGSCs. Its regulatory mechanism may be related to inhibition of phosphorylation of LIF to activate STAT3 and downregulation of NANOG mRNA and protein expression.