NLRP3 inflammasome is an intracellular protein complex of which activation is responsible for maturation of pro-inflammatory cytokines such as interleukin-1beta (IL-1beta) and IL-18. Full activation of NLRP3 inflammasome requires two distinct steps; expression of component proteins as well as pro-IL-1beta induced by pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS), followed by complex formation activated by damage-associated molecular patterns (DAMPs) such as ATP. Although redox-dependent regulation of the complex formation has been suggested, it remains unclear how cellular redox status is regulated during ATP stimulation in cells. Here we describe that glutathione (GSH), an antioxidant peptide abundantly present in cells, plays an important role in ATP-dependent inflammasome activation. LC-MSMS analyses clearly demonstrated that cellular levels of GSH were rapidly decline upon ATP stimulation in LPS-primed mouse macrophage J774.1 cells. Similar levels of GSH were detected in the culture supernatants suggesting that ATP stimulated GSH efflux. It should be noted that ATP treatment also induced efflux of polysulfurated derivatives of GSH such as GSSH and GSSSH as well. P2X7 receptor antagonist A-804598 treatment remarkably inhibited both IL-1beta production as well as decrease of GSH. GSH or its oxidized form GSSG added extracellularly suppressed ATP-dependent decline of intracellular GSH, and more importantly, IL-1beta production. Analyses with use of ROS-sensitive probe DCDHF-DA revealed that ATP stimulation augmented ROS production in macrophages, and it was cancelled in the presence of GSSG. Thus, macrophages use ATP-P2X7 receptor signaling to modulate GSH homeostasis via GSH efflux, thereby regulating redox balance for the inflammasome activation.