Previous studies have implicated a role of G(i) proteins as co-regulators of Toll-like receptor (TLR) activation. These studies largely derived from examining the effect of G(i) protein inhibitors or genetic deletion of G(i) proteins. However, the effect of increased G(i) protein function or G(i) protein expression on TLR activation has not been investigated. We hypothesized that gain of function or increased expression of G(i) proteins suppresses TLR2- and TLR4-induced inflammatory cytokines. Novel transgenic mice with genomic knock-in of a regulator of G protein signaling (RGS)-insensitive Gnai2 allele (G(i2)(G184S/G184S) ; GS/GS) were employed. These mice express essentially normal levels of G(i2) protein; however, the G(i2) is insensitive to its negative regulator RGS thus rendering more sustained G(i2) protein activation following ligand/receptor binding. In subsequent studies, we generated Raw 264.7 cells that stably overexpress G(i2) protein (Raw G(i2)). Peritoneal macrophages, splenocytes, and mouse embryonic fibroblasts (MEF) were isolated from WT and GS/GS mice and were stimulated with LPS, Pam3CSK4, or Poly (I:C). We also subjected WT and GS/GS mice to endotoxic shock (LPS, 25 mg/kg i.p.) and plasma tumor necrosis factor alpha (TNF-) and interleukin (IL)-6 production were determined. We found that in vitro LPS and Pam3CSK4-induced TNF-, and IL-6 production are decreased in macrophages from GS/GS mice compared with WT mice (p<0.05). In vitro, LPS and Pam3CSK4 induced IL-6 production in splenocytes, and in vivo, LPS-induced IL-6 were suppressed in GS/GS mice. Poly (I:C)-induced TNF-, and IL-6 in vitro demonstrated no difference between GS/GS mice and WT mice. LPS-induced IL-6 production was inhibited in MEFs from GS/GS mice similarly to macrophage and splenocytes. In parallel studies, Raw G(i2) cells also exhibit decreased TNF- and IL-6 production in response to LPS and Pam3CSK4. These studies support our hypothesis that G(i2) proteins are novel negative regulators of TLR activation.