The transcription factor NF‐κB has critical functions in biologic responses to genotoxic stimuli. Activation of NF‐κB in response to DNA double strand break (DSB) inducers can be mediated by ATM (ataxia telangiectasia mutated)‐dependent phosphorylation of NEMO (NF‐κB essential modulator). Here, we show that the replication stress inducers hydroxyurea (HU) and aphidicolin also activate this ATM‐dependent signalling pathway. We further show that ATR (ATM‐ and Rad3‐related) interacts with NEMO but surprisingly does not cause NEMO phosphorylation. Consequently, ATR represses NF‐κB activation induced by replication stress. Reduction or increase of ATR expression by RNA interference or overexpression increased or reduced ATM–NEMO association and NF‐κB activation induced by HU. Apoptosis gene expression and chromatin immunoprecipitation analyses indicated that HU and the DSB inducer etoposide caused complex patterns of NF‐κB‐dependent pro‐ and antiapoptotic gene expression with the overall outcome for the former being pro‐apoptotic, whereas the latter antiapoptotic. Thus, replication stress and DSB inducers activate NF‐κB through a conserved pathway with opposite biologic outcomes, and ATR antagonizes ATM function at least in part by competing for NEMO association.