The tunneling nanotube (TNT) is a newly discovered, long and thin tubular structure between cells. In this study, we established a co-culture system for rat primary astrocytes and C6 glioma cells and found that TNTs formed between them. Most of the TNTs initiated from astrocytes towards C6 glioma cells. The formation of TNTs depended on p53. In addition, hydrogen peroxide increased the number of TNTs in the co-culture system. Established TNTs reduced the proliferation of C6 glioma cells. Our data suggest that TNTs between astrocytes and glioma cells facilitate substance transfer and therefore alter the properties, including the proliferation potential, of glioma cells.