The V protein of parainfluenza virus 5 (PIV5) plays an important role in the evasion of host immune responses. The V protein blocks interferon (IFN) signaling in human cells by causing degradation of the STAT1 protein, a key component of IFN signaling, and blocks IFN-beta production by preventing nuclear translocation of IRF3, a key transcription factor for activating IFN-beta promoter. Interleukin-6 (IL-6), along with tumor necrosis factor (TNF)-alpha and IL-1beta, is a major proinflammatory cytokine that plays important roles in clearing virus infection through inflammatory responses. Many viruses have developed strategies to block IL-6 expression. Wild-type PIV5 infection induces little, if any, expression of cytokines such as IL-6 or TNF-alpha, whereas infection by a mutant PIV5 lacking the conserved C-terminal cysteine rich domain (rPIV5VDeltaC) induced high levels of IL-6 expression. Examination of mRNA levels of IL-6 indicated that the transcription activation of IL-6 played an important role in the increased IL-6 expression. Co-infection with wild-type PIV5 prevented the activation of IL-6 transcription by rPIV5VDeltaC, and a plasmid encoding the full-length PIV5 V protein prevented the activation of IL-6 promoter-driven reporter gene expression by rPIV5VDeltaC, indicating that the V protein played a role in inhibiting IL-6 transcription. The activation of IL-6 was independent of IFN-beta even though rPIV5VDeltaC-infected cells produced IFN-beta. Using reporter gene assays and chromatin immunoprecipitation (ChIP), it was found that NF-kappaB played an important role in activating expression of IL-6. We have proposed a model of activating and inhibiting IL-6 transcription by PIV5.