Krüppel-like-factor-5 (KLF5) is a zinc-finger transcription factor involved in cell transformation, proliferation and carcinogenesis. Importantly, KLF5 can be up-regulated by RAS mutations, however, controversy persists whether it functions as a tumor suppressor or an oncogene. Since KRAS is frequently mutated in pancreatic cancer, we investigated the regulation of KLF5 in this cancer entity. Human pancreatic cancer cells (HPAFII, L3.6pl, COLO357, BxPC3, Panc89) as well as the KRAS-mutated colon carcinoma cell line HCT116 were employed. Changes in expression of KLF5 and the effects of KLF5 knock-down were determined by real-time PCR. Effects on signaling pathways were investigated by Western blotting. Effects of cell density on KLF5 expression were investigated by plating cells at various confluences and growing them in full serum media. Three dimensional pancreatic tumor spheroids were generated using the liquid overlay culture technique. KLF5 is over-expressed in pancreatic cancer cells and exceeds KLF5 expression in KRAS-mutated colon cancer cells. In contrast to colon carcinoma cells, blockade of either B-Raf/C-Raf or MAPK/Erk did not reduce the KLF5 levels in pancreatic cancer. Moreover, KLF5 expression levels neither correlated with the mutational status of KRAS, nor with MEK phosphorylation levels in pancreatic cancer cells. Importantly, KLF5 was significantly up-regulated by IL-1β or hypoxic conditions. Inhibition of HIF-1α by RNAi reduced KLF5, whereas the IL-1β-mediated induction of KLF5 was diminished by blocking the p38 pathway. In addition, blocking IL-1 receptor reduced constitutive KLF5 expression. KLF5 was also significantly elevated by high cell density and in tumor spheroids. In conclusion, our study is the first to demonstrate that KLF5 is overexpressed in pancreatic cancer cells and regulated in a different way than in colon cancer cells. Up-regulation of KLF5 in pancreatic cancer may involve the IL-1β/IL-1R system, p38, HIF-1α and cell contact.