The proproliferative transcription factor KLF5 plays an important role in promoting cell proliferation and tumorigenesis. KLF5 is a short-lived protein that can be rapidly degraded through the ubiquitin-proteasome pathway in cancer cells. However, the mechanisms regulating protein stability remain poorly understood. In this study, the tumor suppressor Fbw7, a component of the SCF complex (SCF(Fbw7)) E3 ubiquitin ligase, specifically promoted the degradation and ubiquitination of KLF5 but had little effect on the stability of KLF4. Fbw7 interacted with KLF5 in a CDC4 phosphodegron (CPD)-dependent manner. Three CPDs were found in the KLF5 protein. Simultaneous mutation of these CPDs significantly abolished Fbw7-mediated ubiquitination and degradation. Furthermore, Fbw7 deficiency dramatically delayed KLF5 turnover and led to the accumulation of KLF5 protein in cancer cells. Glycogen synthase kinase-3beta could phosphorylate and promote Fbw7-mediated KLF5 degradation. More importantly, Fbw7 negatively regulated the biological activity of KLF5 in gene regulation and cell proliferation. Taken together, these data indicate that Fbw7 is a key negative regulator controlling KLF5-mediated cell proliferation and suggest an additional mechanism linking the loss of Fbw7 function to tumorigenesis.