Although Kruppel-like factor 5 (KLF5) is a transcription factor that has been implicated in pathways critical to carcinogenesis, controversy persists as to whether it functions as a tumor suppressor or as an oncogene. Here, we describe a novel role for KLF5 in a p53-independent apoptotic pathway. Using RNA-interference technology, we show that cells deficient in KLF5 have increased sensitivity to DNA damage, regardless of p53 status. Both p53 and p53-dependent factors are unaffected by KLF5 depletion. Instead, the apoptotic phenotype consequent to damage is associated with reduced bad phosphorylation, and downregulation of Pim1. Consistently, transfection of wild-type Pim1 is sufficient to rescue this phenotype. Previous data have shown a number of putative Sp1-binding consensus sequences on the Pim1 promoter. Remarkably, chromatin immunoprecipitation studies show that KLF5 binds to the Pim1 promoter, and that binding increases soon after damage. These results identify a novel, p53-independent apoptotic pathway through which KLF5 functions in response to DNA damage. Therapeutic deregulation of this pathway could be used to modulate chemosensitivity. © 2008 Nature Publishing Group All rights reserved.