Human respiratory syncytial virus (HRSV) is a member of the family Paramyxoviridae, and is responsible for serious respiratory illness in infants, the elderly and the immunocompromised. HRSV exists as two distinct lineages known as subgroups A and B, which represent two lines of divergent evolution with extensive genetic and serologic differences. While both subgroup A and B viruses contribute to overall HRSV disease, subgroup A isolates are associated with both increased frequency and morbidity of infections, and reasons for this are unclear. HRSV disease is characterized by virus-mediated cell destruction in combination with extensive inflammatory and immune modulatory responses, and for HRSV subgroup A isolates, several of these signaling pathways are regulated through activation of the transcription factor NF-κB. In contrast, the NF-κB activation characteristics of HRSV subgroup B infection remain untested. Here, we performed a quantitative and comparative analysis of NF-κB activation in response to infection of both continuous and primary cell cultures with HRSV subgroup A and B isolates. Our results showed the model HRSV subgroup A isolate consistently induced increased NF-κB activation compared to its HRSV subgroup B counterpart. The differential NF-κB activation characteristics of HRSV subgroup A and B viruses may contribute to differences in their pathogenesis.