Krüppel-like factor 5 (KLF5) is a transcription factor involved in cell transformation, proliferation, and carcinogenesis that can be up-regulated by RAS mutations. However, controversy persists as to whether it functions as a tumor suppressor or as an oncogene. Because KRAS is frequently mutated in pancreatic cancer, we investigated the regulation of KLF5 in this cancer entity. Our results show that KLF5 is overexpressed in pancreatic cancer cells and exceeds KLF5 expression of KRAS-mutated colon cancer cells. Surprisingly, inhibition of B-Raf/C-Raf or MAPK/Erk did not reduce KLF5 levels, suggesting that KLF5 expression is not promoted by KRAS-Raf-MEK-Erk signaling in pancreatic cancer. This finding is in striking contrast to reports on MEK-Erk-mediated KLF5 induction in colon cancer cells. Moreover, KLF5 expression levels neither correlated with the mutational status of KRAS nor with MEK phosphorylation in pancreatic cancer cells. Importantly, KLF5 was significantly up-regulated by interleukin (IL)-1beta or hypoxia. The IL-1 beta-mediated induction of KLF5 was diminished by blocking the p38 pathway. In addition, blocking IL-1R reduced the constitutive KLF5 expression, suggesting an autocrine activation loop. Moreover, KLF5 coimmunoprecipitated with hypoxia-inducible factor-1alpha (HIF-1alpha) and HIF-1alpha(siRNA) reduced constitutive KLF5. Similarly, KLF5(siRNA) reduced the expression of the HIF-1alpha target gene GLUT-1. Furthermore, KLF5 expression was significantly elevated by high cell density, by anchorage-independent cell growth, and in tumor spheroids. Down-regulation of KLF5 by RNAi reduced the expression of the target genes, survivin, and platelet-derived growth factor-A. In conclusion, overexpression of KLF5 in human pancreatic cancer cells is not mediated by KRAS/Raf/MAPK/Erk signaling, but involves the IL-1beta/IL-1R system, p38, and the transcription factor HIF-1alpha.