Although the zinc finger transcription factor Krüppel-like factor 4 (KLF4) has been shown to be a negative regulator of cell proliferation, the mechanisms underlying the posttranslational modification of KLF4, especially at the level of protein degradation, are poorly understood. Here, we show that KLF4 protein levels in quiescent cells were high, but decreased rapidly as cells entered the proliferating stage following serum stimulation. This decrease was partially reversed by pretreatment with MG132, a proteasome inhibitor. Moreover, KLF4 was an unstable protein that underwent rapid turnover, and exhibited a relatively short half-life (t1/2 approximately 120 minutes). To investigate the involvement of the ubiquitin-proteasome pathway in the regulation of the stability of KLF4, HCT116 cells were treated with proteasome inhibitors. Our results showed that, following lactacystin treatment, levels of endogenous KLF4 increased in a time- and dose-dependent manners. Using a cell-free system, in vitro-translated 35S-labeled KLF4 protein was degraded by protein extracts prepared from exponentially growing HCT116 cells in the presence of ATP. These effects were prevented by pretreatment with MG132 or replacement of ATP with ATP-gamma-S, a nonhydrolyzable analogue of ATP, suggesting that ATP is required for KLF4 degradation by the 26S proteasome. In addition, KLF4 was subject to ubiquitination when cells were treated with the proteasome inhibitor or transfected with exogenous ubiquitin. Collectively, these results indicate that destabilization of KLF4 following serum stimulation is mediated, at least in part, through a ubiquitin-proteasome pathway.