Mounting studies have revealed that long non-coding RNA (lncRNA) deleted in lymphocytic leukemia 1 (DLEU1) positively regulated the initiation and development of various human malignant tumors. Nevertheless, the function and mechanism of DLEU1 in human glioblastoma multiforme (GBM) remain elusive and ill-defined. The current study was designed to highlight the functional role and disclose the underlying molecular mechanism by which DLEU1 regulated GBM development. We found that DLEU1 was up-regulated in GBM and DLEU1 knockdown significantly inhibited GBM cell proliferation and induced apoptosis. As predicted by bioinformatics analysis and validated in mechanistic assays, SP1 could bind to the promoter region of DLEU1 to activate DLEU1 transcription. Additionally, miR-4429 was verified as a target gene of DLEU1 and negatively modulated by DLEU1. More importantly, miR-4429 overexpression repressed the mRNA and protein levels of SP1 via binding to the 3′UTR of SP1. Overexpression of SP1 or miR-4429 inhibitor could partly abolish the effect of DLEU1 knockdown on cell viability and apoptosis in GBM. Accordingly, our experimental data revealed that SP1–DLEU1–miR-4429 formed a feedback loop to promote GBM development, providing a new evidence for the role of DLEU1 in GBM.