Toll-like receptors (TLRs) family may play important roles in inflammatory bowel disease. This study examined the expression of TLR2, TLR4 and TLR9 in the colonic tissues of patients with ulcerative colitis (UC) and explored their roles in the pathogenesis of UC. Colonic biopsies were taken from the colon of 30 patients with mild or moderate UC (at active phase) and 10 healthy controls during colonoscopy. TLR2, TLR4 and TLR9 protein expression levels were immunohistochemically detected. The mRNA expression levels of TLR2, TLR4 and TLR9 were assessed by reverse transcription polymerase chain reaction (RT-PCR). The disease activity index (DAI), colonoscopic and histologic grades and fecal microbial flora were determined. Histological examination showed that the intestinal mucous membrane of UC patients underwent acute inflammation changes. Immunohistochemistry exhibited that the expression levels of TLR2, TLR4 and TLR9 in colon epithelia and inflammatory cells were higher in UC patients than in control group (P<0.01). The mRNA expression levels of TLR2, TLR4 and TLR9 were increased in UC patients but were not detected in the normal controls. Expression levels of TLR2, TLR4 and TLR9 were positively correlated, and bore close correlation with DAI, colonoscopic and histologic grades and fecal microbial flora. An important mechanism of UC might be that abnormal activation of mucosal immunity by intestinal dysbacteriosis caused dysregulation of TLRS that mediates innate immunity.