KAISO, a member of the POK protein family, is induced by DNA-damaging agents to enhance apoptosis in a p53-dependent manner. Previously, we found that p53 interacts with KAISO, and acetylation of p53 lysine residues by p300 is modulated by KAISO. APAF1, the core molecule of the apoptosome, is transcriptionally activated by KAISO only in cells expressing p53, which binds to APAF1 promoter p53-response elements (p53REs). APAF1 transcriptional upregulation is further enhanced by KAISO augmentation of p53 binding to the APAF1 promoter distal p53RE#1 (bp, -765 to -739). Interestingly, a NF-κB response element, located close to the p53RE#1, mediates APAF1 transcriptional repression by affecting interaction between KAISO and p53. Ectopic RelA/p65 expression led to depletion of nuclear KAISO, with KAISO being mainly detected in the cytoplasm. RelA/p65 cytoplasmic sequestration of KAISO prevents its nuclear interaction with p53, decreasing APAF1 transcriptional activation by a p53-KAISO-p300 complex in cells exposed to genotoxic stresses. While KAISO enhances p53-dependent apoptosis by increasing APAF1 gene expression, RelA/p65 decreases apoptosis by blocking interaction between KAISO and p53. These findings have relevance to the phenomenon of cancer cells' diminished apoptotic capacity and the onset of chemotherapy resistance.