KLF6 is an evolutionary conserved and ubiquitously expressed mammalian transcription factor that has been involved in cell cycle control mechanisms in normal and cancer cells. This work aimed at characterizing the role of KLF6 in apoptosis regulation in response to DNA-damaging agents commonly used in anticancer therapies. Here, we report that during apoptosis triggered by different DNA-damaging drugs the KLF6 protein levels were drastically downregulated in Cos-7 and HepG2 cells. Moreover, we showed during apoptosis a strong downregulation of the KLF6 mRNA and its transcriptional activity determined by Real-Time PCR and reporter assays, respectively. Additional studies established that KLF6 was silenced in apoptotic cells after DNA damage in a p53-independet manner. Thus, the absence of the KLF6 protein proposes that it might be an inhibitor of apoptosis-induced by DNA-damaging drugs. To test this possibility RNA interference was successfully employed to knockdown KLF6 in HepG2 cells by using small interfering RNAs (siRNAs) targeted to different regions of KLF6 mRNA. Interestingly, KLF6 loss by siRNA sensitized cells to apoptosis triggered by DNA-damaging agents. Altogether our results in which KLF6 protein levels decreased during DNA damage induced apoptosis and that siRNA-mediated loss of KLF6 favored cell death, support the notion that KLF6 behaves as an anti-apoptotic factor.