AbstractNumerous studies have reported that Vpr alters NF‐κB signaling in various cell types, however, the findings have been largely conflicting with reports of both stimulatory and inhibitory effects of Vpr. Our aim was to investigate the role of Vpr signaling in myeloid cells using an adenovirus based expression and indicator system. Our results show that Vpr is inhibitory to NF‐κB, however, this effect is dependent on the particular manner of NF‐κB stimulation. Consistent with this notion, we report that Vpr has inhibitory effects that are specific to the TNF‐α pathway, but not affecting the LPS pathway, suggesting that differential targets of Vpr may exist for NF‐κB regulation. Further, we identify VprBP as one possible cellular component of Vpr's regulation of IκBα in response to TNF‐α stimulation. We did not identify such a role for HSP27, which instead seems to inhibit Vpr functions. Chronically HIV‐1 infected U1 cells with knockdown constructs for Vpr were unexpectedly less responsive to TNF‐α mediated viral replication, perhaps suggesting that other HIV‐1 components may antagonize these anti‐NF‐κB effects in infected cells. We hypothesize that Vpr may serve an important role in the context of viral infection and immune function in vivo, through its selective inhibition of NF‐κB pathways. J. Cell. Physiol. 228: 781–790, 2013. © 2012 Wiley Periodicals, Inc.