Gliomas are the most common primary brain tumors in the central nervous system and a leading cause of tumor-related death. High-mobility group nucleosome binding domain 5 (HMGN5/NSBP1), which is highly expressed in breast cancer and in hormone-induced mouse uterine adenocarcinoma, acts as a potential oncogene in gliomas. In this study, the role of HMGN5 in the proliferation of human glioma cells was investigated by lentivirus-mediated RNA interference (RNAi). The decrease in HMGN5 expression in human glioma U251 and U87 cells caused cell cycle arrest in the G1 phase and a delay in cell proliferation, as well as resulting in more apoptosis and an inhibition of clonogenic growth in soft agar in U251 cells; these results suggest that HMGN5 is required for tumorigenesis in vitro. Furthermore, HMGN5 was highly expressed in both high-grade and low-grade glioma tissue samples compared with normal brain tissues. Collectively, our data suggest that HMGN5 may play a critical role in the development of gliomas.