Heat shock inhibits NF-κB signaling, yet the knowledge about its influence on the regulation of NF-κB-dependent genes is limited. Using genomic approaches, i.e., expression microarrays and ChIP-Seq, we aimed to establish a global picture for heat shock-mediated impact on the expression of genes regulated by TNFα cytokine. We found that 193 genes changed expression in human U-2 osteosarcoma cells stimulated with cytokine (including 77 genes with the κB motif in the proximal promoters). A large overlap between sets of genes modulated by cytokine or by heat shock was revealed (86 genes were similarly affected by both stimuli). Binding sites for heat shock-induced HSF1 were detected in regulatory regions of 1/3 of these genes. Furthermore, pre-treatment with heat shock affected the expression of 2/3 of cytokine-modulated genes. In the largest subset of co-affected genes, heat shock suppressed the cytokine-mediated activation (antagonistic effect, 83 genes), which genes were associated with the canonical functions of NF-κB signaling. However, subsets of co-activated and co-repressed genes were also revealed. Importantly, pre-treatment with heat shock resulted in the suppression of NF-κB binding in the promoters of the cytokine-upregulated genes, either antagonized or co-activated by both stimuli. In conclusion, we confirmed that heat shock inhibited activation of genes involved in the classical cytokine-mediated functions of NF-κB. On the other hand, genes involved in transcription regulation were over-represented in the subset of genes upregulated by both stimuli. This suggests the replacement of NF-κB-mediated regulation by heat shock-mediated regulation in the latter subset of genes, which may contribute to the robust response of cells to both stress conditions.