Differentiation of glioblastoma stem cells (GSCs) may lead to inhibition of their self-renewing ability and tumorigenic potential, as well as increasing their sensitivity to treatment. The critical role of long non-coding RNAs (lncRNAs) in numerous biological processes has been revealed. However, the involvement of lncRNAs in GSC differentiation remains to be elucidated. In the present study, GSCs were isolated from patient samples and differentiation was induced. Using a high-throughput microarray, the present study identified a profile of 1,545 lncRNAs and 2,729 mRNAs that differed between GSCs and their non-differentiated counterparts. To ascertain the association between the altered lncRNAs and mRNAs, a co-expression network was constructed in which 1,087 lncRNAs and 1,928 mRNAs altered upon GSC differentiation formed a total of 19,642 lncRNA-mRNA pairs. Based on the co-expression network, the lncRNA functions were additionally predicted by a cis- or trans- targeting program. Furthermore, three pairs of lncRNAs and their nearby target mRNAs were selected [ENSG00000261924.1-regulatory associated protein of MTOR complex 1, ENSG00000235427.1-caveolin 1 and Tax1 binding protein 3 (TAX1BP3)-purinergic receptor P2X 5 (P2RX5)-TAX1BP3] and their expression levels were validated by reverse transcription-quantitative polymerase chain reaction. The altered lncRNAs were also regulated by various pluripotency transcription factors (POU domain, class 3, transcription factor, sex determining region Y-box 2, spalt-like transcription factor 2 and oligodendrocyte lineage transcription factor 2). In conclusion, the results of the present study revealed that lncRNAs may function in GSC differentiation by regulating their target mRNAs, and a set of lncRNAs were identified as candidates for further study concerning the future treatment of GSCs.