BACKGROUND: We have previously shown that the transcriptional coregulator NCoR represses astrocytic differentiation of neural stem cells, suggesting that NCoR could be a plausible target for differentiation therapy of glioblastoma. METHODS: To study a putative role for NCoR in regulating glioblastoma cell characteristics, we used RNA-mediated knockdown followed by analysis of gene expression, proliferation and cell growth, autophagy, invasiveness in vitro, and tumor formation in vitro and in vivo. We further performed chromatin immunoprecipitation of NCoR followed by genome-wide sequencing in the human glioblastoma cell line U87 in order to reveal NCoR-occupied loci. RESULTS: RNA knockdown of NCoR resulted in a moderate increase in differentiation accompanied by a significant decrease in proliferation in adherent U87 human glioblastoma cells. chromatin immunoprecipitation sequencing approach revealed alternative mechanisms underlying the decrease in proliferation, as NCoR was enriched at promoters of genes associated with autophagy such as ULK3. Indeed, signs of an autophagy response in adherent glioblastoma cells included an increased expression of autophagy genes, such as Beclin1, and increased lipidation and nuclear puncta of LC3. Intriguingly, in parallel to the effects in the adherent cells, NCoR knockdown resulted in a significant increase in anchorage-independent growth, and this glioblastoma cell population showed dramatic increases in invasive properties in vitro and tumor formation capacity in vitro and in vivo along with an increased proliferation rate. CONCLUSION: Our results unveil unexpected aspects of NCoR regulation of tumor characteristics in glioblastoma cells and highlight the need for caution when transposing developmental concepts directly to cancer therapy.