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How macrophages maintain redox homeostasis in the inflammatory process, in which a large amount of oxidants are produced, remains elusive. In this study, we investigated the temporal changes in the intracellular glutathione (GSH), the master antioxidant, and the expression of glutamate cysteine ligase (GCL), the rate-limiting enzyme for GSH biosynthesis, in the inflammatory response of human macrophages (THP1 cells) to lipopolysaccharide. Intracellular GSH concentration was decreased significantly in the early phase (˜6 h) of LPS exposure, and then gradually went back to the basal level in the late phase (9–24 h). The expression level of the catalytic subunit of GCL (GCLC) followed a similar pattern of change as GSH: its mRNA and protein levels were reduced in the early phase and then back to basal level in the late phase. In contrast, the expression of the modifier subunit of GCL (GCLM) was significantly increased in the phase of LPS exposure. Activation Nrf2, the transcription factor involved in the induction of both GCLC and GCLM, occurred at as early as 3 h after LPS exposure; whereas the activation of NF-κB occurred at as early as 30 min. Inhibition of NF-κB signaling with SN50 prevented the decrease of GCLC and inhibited Nrf2 activation in response to LPS. These data demonstrate time-dependent changes in the expression of GCL and Nrf2 signaling during the inflammatory response, and that the regulation of GCLC and GCLM might be through different pathways in this process.GSH concentration altered kinetically in macrophages in response to LPS.GSH decrease in the early phase of LPS exposure coincided with a decreased expression of GCLC.NF-κB inhibition prevented LPS-caused decrease in GCLC but did not affect LPS-induced GCLM.NF-κB inhibition abrogated LPS-mediated Nrf2 activation in macrophages.