The stress inducible transcription factor, NF-κB induces genes involved in proliferation and apoptosis. Aberrant NF-κB activity is common in cancer and contributes to therapeutic-resistance. Poly(ADP-ribose) polymerase-1 (PARP-1) is activated during DNA strand break repair and is a known transcriptional co-regulator. Here, we investigated the role of PARP-1 function during NF-κB activation using p65 siRNA, PARP siRNA or the potent PARP-1 inhibitor, AG-014699. Survival and apoptosis assays showed that NF-κB p65 −/− cells were more sensitive to ionizing radiation (IR) than p65 +/+ cells. Co-incubation with p65 siRNA, PARP siRNA or AG-014699 radio-sensitized p65 +/+, but not p65 −/− cells, demonstrating that PARP-1 mediates its effects on survival via NF-κB. Single strand break (SSB) repair kinetics, and the effect SSB repair inhibition by AG-014699 were similar in p65 +/+ and p65 −/− cells. Since preventing SSB repair did not radio-sensitize p65 −/− cells, we conclude that radio-sensitization by AG-014699 is due to downstream inhibition of NF-κB activation, and independent of SSB repair inhibition. PARP-1 catalytic activity was essential for IR-induced p65 DNA binding and NF-κB-dependent gene transcription, whereas for TNF-α treated cells, PARP-1 protein alone was sufficient. We hypothesize that this stimulus-dependent differential is mediated via stimulation of the Poly(ADP-ribose) polymer, which was induced following IR, not TNF-α. Targeting DNA-damage activated NF-κB using AG-014699 may therefore overcome toxicity observed with classical NF-κB inhibitors without compromising other vital inflammatory functions. These data highlight the potential of PARP-1 inhibitors to overcome NF-κB-mediated therapeutic resistance and widens the spectrum of cancers in which these agents may be utilized.