Mitochondria are increasingly recognized as key regulators of immune cell function, but it is unclear how their reprogramming contributes to pathogen specific responses. Using a combination of immunological and biochemical techniques, this study investigated the potential role of the transcription factor hypoxia-inducible factor-1α (HIF-1α) in driving inflammatory cytokine production in antiviral immune responses.  This transcription factor has been shown to regulate mitochondrial function, oxygen consumption and substrate choices to support energy metabolism We found several mechanisms, including mitochondrial reactive oxygen species, contribute to the stabilization of HIF-1α in macrophages activated by viral ligands. This stabilization results in the accumulation of HIF-1α, which plays a central role in amplifying inflammatory cytokine production. Further, we found this amplification occurs independently of HIF’s transcriptional activity and instead was associated with the ability of HIF-1α to directly modulate NF-κB activity. This study shows for the first time that HIF-1α serves to fine tune the level of inflammatory cytokine production independently of its transcriptional activity during antiviral immune responses and that it may act to drive differential responses to varying tissue microenvironments. It also suggests that HIF-1α and the proteins/molecules that stabilize its accumulation may represent potential targets to reduce potentially damaging inflammation during chronic viral infections or in other contexts where antiviral immune responses become chronically activated.