The lipopolysaccharide (LPS)–induced endocytosis of Toll-like receptor 4 (TLR4) is an essential step in the production of interferon-β (IFN-β), which activates the transcription of antiviral response genes by STAT1 phosphorylated at Tyr⁷⁰¹. Here, we showed that STAT1 regulated proinflammatory cytokine production downstream of TLR4 endocytosis independently of IFN-β signaling and the key proinflammatory regulator NF-κB. In human macrophages, TLR4 endocytosis activated a noncanonical phosphorylation of STAT1 at Thr⁷⁴⁹, which subsequently promoted the production of interleukin-6 (IL-6) and IL-12p40 through distinct mechanisms. STAT1 phosphorylated at Thr⁷⁴⁹ activated the expression of the gene encoding ARID5A, which stabilizes IL6 mRNA. Moreover, STAT1 phosphorylated at Thr⁷⁴⁹ directly enhanced transcription of the gene encoding IL-12p40 (IL12B). Instead of affecting STAT1 nuclear translocation, phosphorylation of Thr⁷⁴⁹ facilitated the binding of STAT1 to a noncanonical DNA motif (5′-TTTGANNC-3′) in the promoter regions of ARID5A and IL12B. The endocytosis of TLR4 induced the formation of a complex between the kinases TBK1 and IKKβ, which mediated the phosphorylation of STAT1 at Thr⁷⁴⁹. Our data suggest that noncanonical phosphorylation in response to LPS confers STAT1 with distinct DNA binding and gene-regulatory properties that promote both IL12B expression and IL6 mRNA stabilization. Thus, our study provides a potential mechanism for how TLR4 endocytosis might regulate proinflammatory cytokine production.