IRAK3 is a critical negative regulator of innate immunity, and involved in many diseases such as sepsis and asthma. Thus, IRAK3 is potentially a diagnostic biomarker or immunotherapy target. IRAK3 down-regulates inflammatory responses and is required for endotoxin tolerance where the production of inflammatory cytokines is diminished upon constant exposure or re-challenge of endotoxins. However, mechanisms of IRAK3 actions are not fully understood, and IRAK3 actions in inflammation are dependent on cell type, type of stimuli and stimulation duration. Recent studies showed IRAK3 contains a guanylate cyclase (GC) centre that can generate cyclic guanosine monophosphate (cGMP) (1). Mutations in the catalytic site of the GC centre modify IRAK3 function in transiently transfected HEK293 cells (1). cGMP is a second messenger regulating multiple physiological processes including inflammation. Since effects of low levels of cGMP on inflammation are unknown, we undertook dose-response studies on monocytic cell lines (THP-1 and THP-1 BLUE) during endotoxin challenge measuring inflammatory responses. Sub-nanomolar concentrations (0.1 nM) of membrane permeable 8-Br-cGMP reduced LPS-induced NF-κB activity and cytokine (IL-6 and TNF-α) production. Pharmacologically upregulated cellular cGMP level reduced cytokine production. Downregulation of cellular cGMP increased the level of cytokines, while membrane permeable cGMP at 0.1 nM inhibited this effect. Using CRISPR/Cas9 we generated IRAK3 knock-down THP-1 cell lines to investigate if IRAK3 was necessary for cGMP effects on inflammation. Unlike the wildtype cells, 8-Br-cGMP at 0.1 nM to 100 nM did not suppress inflammatory responses in IRAK3-knockdown cells. Complementation of the IRAK3-knockdown cells with wildtype IRAK3, but not mutant IRAK3, suppressed cytokine production. Taken together, low levels of cGMP form a critical component of IRAK3 action and that this indeed may be via cGMP enriched nanodomain formed by IRAK3 itself.