Objective To investigate the correlation between Toll-like receptor 4 (TLR4) signal and the development and progression of ulcerative colitis (UC). Methods The colon biopsy samples (n＝63) were collected from UC patients and were divided into Matts’ histological grade 1-5. The TLR4 expression level was detected by immunohistochemistry. TLR4-immunoglobulin (Ig) fusion protein was prepared using FreeStyle 293 expression system. Peripheral blood mononuclear cells (PBMCs) from healthy individuals were cultured in vitro, 100 μg/mL lipopolysaccharide was added to induce inflammation, and 100 μg/mL TLR4-Ig was added to block TLR4. Then the secretion levels of 10 different inflammatory cytokines, including granulocyte-macrophage colony-stimulating factor, interferon γ, interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, and tumor necrosis factor α, were detected. The acute colitis mouse model was induced by dextran sulfate sodium (DSS), and was intervened with 20 mg/kg TLR4-Ig, 40 mg/kg TLR4-Ig or antibiotics＋40 mg/kg TLR4-Ig, respectively. Then the colon length of mice was measured and histopathological evaluation was performed. Results The expression level of TLR4 in colon tissues of UC patients was positively correlated with Matts’ histological grades (P＜0.001). TLR4-Ig fusion protein could bind TLR4 ligands with high affinity, blocking the activation of PBMCs and the secretion of inflammatory cytokines mediated by TLR4 signal. The blocking of TLR4 signal by TLR4-Ig fusion protein aggravated DSS-induced acute colitis in mice, and treatment with antibiotics alleviated the aggravation of the colitis caused by TLR4 signal blocking. Conclusion The communication between TLR4 signal and intestinal flora is an important protective mechanism at the early stage of UC. The absence of TLR4 signal is not conducive to relieving acute inflammation and repairing intestinal mucosa.