G protein-coupled receptor (GPCR) kinase 2 (GRK2) has emerged as an integrating node in many signaling pathways besides its GPCR signaling role. We investigated how GRK2 could contribute to Toll-like receptor (TLR4) signaling for iNOS expression in microglia. We found in microglial cells LPS-induced iNOS expression requires both STAT1 and STAT3 activation. GRK2 knockdown by siRNA transfection suppressed phosphorylation and nuclear translocation of STAT1/3. Interferon-β (IFN-β) produced from TLR4 signaling induce phosphorylation of STAT1/3, and its expression is regulated by interferon regulatory factors (IRF) at transcription level. We found that GRK2 knockdown suppressed LPS-induced IFN-β production and mRNA expression. LPS stimulation induce IRF1 expression and nuclear translocation, and GRK2 knockdown suppressed IRF1 nuclear translocation without affecting expression level. These results suggest that GRK2 positively regulates IRF1 activation to induce IFN-β expression, STAT1/3 activation, and subsequently iNOS expression. Furthermore, GRK2 knockdown suppressed exogenous IFN-β-induced STAT1/3 activation. In conclusion, GRK2 upregulates TLR4-induced iNOS expression by mediating IRF1 and STAT1/3 activation.