Aims: S-nitrosylation and S-glutathionylation, redox-based modifications of protein thiols, are recently emerging as important signaling mechanisms. In this study, we assessed S-nitrosylation-based regulation of Janus-activated kinase 2/signal transducer and activator of transcription 3 ( JAK2/STAT3) pathway that plays critical roles in immune/inflammatory responses and tumorigenesis. Results: Our studies show that STAT3 in stimulated microglia underwent two distinct redox-dependent modifications, S-nitrosylation and S-glutathionylation. STAT3 S-nitrosylation was associated with inducible nitric oxide synthase (iNOS)-produced nitric oxide (NO) and S-nitrosoglutathione (GSNO), whereas S-glutathionylation of STAT3 was associated with cellular oxidative stress. NO produced by iNOS or treatment of microglia with exogenous GSNO inhibited STAT3 activation via inhibiting STAT3 phosphorylation (Tyr 705 ). Consequently, the interleukin-6 (IL-6)-induced microglial proliferation and associated gene expressions were also reduced. In cell-free kinase assay using purified JAK2 and STAT3, STAT3 phosphorylation was inhibited by its selective preincubation with GSNO, but not by preincubation of JAK2 with GSNO, indicating that GSNO-mediated mechanisms inhibit STAT3 phosphorylation through S-nitrosylation of STAT3 rather than JAK2. In this study, we identified that