Glioma is the most common sub-type of brain tumor. Due to the presence of stem-like cells, it is characterized by poor prognosis, aggressive ability and high post-surgical recurrence rates. Hence, there is critical need to identify molecular mechanisms of glioma stem-like cells. We found a novel lncRNA in the ZNF281 gene and named it lncRNA-ZNF281. We detected the expression of lncRNA-ZNF281 in glioma stem-like cells (U251s), the glioma cell line (U251) and also in normal brain tissue. The expression of lncRNA-ZNF281 was lower in glioma stem-like cells (U251s) and this indicates that lncRNA-ZNF281 can regulate the self-renewal capacity of glioma stem-like cells and stem cell marker expression. Most significantly, lncRNA-ZNF281 inhibits the invasion of glioma stem-like cells by regulating the expression of the NF-κB1 signaling pathway. Our data demonstrates that lncRNA-ZNF281 inhibits the self-renewing ability and invasion of GSCs in vitro and in vivo and can reduce tumorigenicity in the glioma stem-like cell (U251s). The underlying mechanisms may involve the regulation of stem cell markers (CD133, Nestin, OCT4 and Nanog) to reduce the self-renewal ability and regulate the NF-κB1 signaling pathway and inhibit U251s glioma stem-like cell invasion. These finding suggest that lncRNA-ZNF281 could be a successful new therapeutic target in glioma.