Krüppel-like factor 4 (KLF4) is an evolutionarily conserved zinc finger-containing transcription factor with diverse regulatory functions in cell growth, proliferation, differentiation, and embryogenesis. However, little is known about the response of KLF4 to heat stress. In this study, Western blot and reverse transcriptase-polymerase chain reaction were performed to determine the changes in KLF4 expression in response to heat stress. The results showed that heat stress up-regulated KLF4 messenger RNA and protein levels in a time-dependent manner in vivo and in 4 cell lines. Moreover, a study with heat shock transcription factor 1 (Hsf1) gene knockout mice indicated that the induction of KLF4 in response to heat stress was mediated by Hsf1. This process occurred rapidly, indicating that KLF4 is an immediate early response gene of heat stress. Next, the roles of KLF4 under heat stress conditions were analyzed for cells overexpressing or deficient in KLF4. The results showed overexpression of KLF4 increased the death rate of C2C12 cells, whereas KLF4 deficiency decreased the injury of C2C12 cells from heat stress conditions, suggesting that KLF4 might play an important role in cell injury induced by heat stress. KLF4 might be an immediate early response gene and could play an important role in cell injury induced by heat stress.