Significance Inflammation is often accompanied by temperature change, but little is known about the role of temperature in the inflammatory response. We show that physiologically relevant temperature changes significantly perturb NF-κB dynamics following TNFα stimulation in single cells. Using experimentation informed by mathematical modeling, we found that these changes were mediated, at least in part, through the key feedback gene TNFAIP3/A20. Curtailing A20 expression removed temperature sensitivity across the fever range (37 °C to 40 °C). Gene expression was generally unaffected between these temperatures, although a select set of NF-κB−regulated genes was up-regulated at early time points. These genes were predominantly involved in inflammation, signaling, and cell fate. The cellular response to inflammation may therefore be mechanistically and functionally regulated by temperature.