Although both the heat shock protein 70 (HSP70) and the activating transcription factor 5 (ATF5) have been shown to promote cell survival of transformed cells but not survival of non-transformed cells, the relationship of the two molecules is unknown. Here we show that HSP70 and ATF5 are concomitantly up-regulated upon transient but down-regulated over prolonged cellular stress and apoptotic stimulation in the rat C6 glioma and human U87 glioma cells. HSP70 interacts strongly with the N-terminal activation domain of ATF5, which is expected to be rigid and uniquely structured under physiological conditions because of extraordinary high concentration (over 25%) of proline residues. Binding of HSP70 to ATF5 is an ATP-driven process and requires functional ATPase on the nucleotide binding domain of the HSP70 molecule. Overexpression of HSP70 dramatically stabilizes the ATF5 protein, which is otherwise subject to rapid degradation, facilitated by both proteasome-dependent and caspase-dependent processes, whereas HSP70 depletion leads to acceleration of ATF5 degradation and transcription repression of Bcl-2 and Egr-1, which are downstream targets of ATF5 in C6 and U87 glioma cells. Our data reveal an essential role for HSP70 in maintaining high levels of ATF5 expression in glioma cells and support the conclusion that ATF5 is an important substrate protein of HSP70 that mediates HSP70-promoted cell survival in glioma cells.