Lipopolysaccharide (LPS)–induced toll-like receptor 4 (TLR4) endocytosis has emerged as a key step for the production of interferon (IFN)-β, which activates the transcription of antiviral response genes through Janus kinase (JAK)/pTyr701 signal transducer and activator 1 (STAT1) signaling. TLR4 endocytosis also promotes proinflammatory cytokines production, at least in part through mediating a late-phase of nuclear factor (NF)-κB activation. However, NF-κB activation alone cannot explain the full spectrum of how TLR4 endosomal signaling conduits the production of proinflammatory cytokines. Our study identified STAT1 as a proinflammatory effector downstream of TLR4 endocytosis independent of IFN-β signaling or NF-κB activity. In human macrophages, TLR4 endocytosis activates noncanonical phosphorylation of STAT1 at Thr749 (pThr749), which subsequently promotes the proinflammatory response rather than the IFN response. pThr749 STAT1 prolongs the half-life of interleukin (IL)-6 mRNA through activating the transcription of AT-rich interactive domain-containing protein 5A (ARID5A), which stabilizes IL-6 mRNA. Furthermore, pThr749 STAT1 promotes a late-phase of the transcription of IL-12. We demonstrated that pThr749 confers STAT1 with distinct gene-regulatory prosperities and facilitates STAT1 binding to a noncanonical DNA motif (5’...TTTGANNC...3’) at the promoter regions of ARID5A and IL-12. Our results indicate that different phosphorylation of STAT1 confers distinct DNA binding and gene regulation downstream of TLR4 endocytosis where pTyr701 promotes the IFN response while pThr749 promotes the proinflammatory response. By unveiling an alternative activation of STAT1, our study adds another piece to the puzzle of how TLR4 endocytosis regulates the production of proinflammatory cytokines, which may help in developing biologics or diagnostics for proinflammatory diseases.