Increased hyaluronan (HA) deposition is a common feature of inflamed tissues, including inflammatory bowel disease (IBD)-involved intestines. However, whether HA accumulation promotes or is the result of intestinal inflammation is unknown. Using the mouse dextran sulfate sodium (DSS)-induced experimental model of colitis, we investigated changes in HA deposition in the colon over time in conjunction with evolving pathological changes of tissue architecture. Profound changes in colon HA deposition occurred within 3-7 days of oral DSS administration and, more important, they preceded the inflammatory infiltrate. Interestingly, HA deposition within blood vessels of the colon is observed as early as 3 days during the course of colitis induction, well before any significant inflammatory infiltrate. HA deposition is also observed in blood vessels of inflamed human colon of IBD patients. We determined that human intestinal endothelial cells generate HA in response to proinflammatory stimuli by demonstrating a TNF-alpha-induced increase in hyaluronan synthase-3 mRNA expression and the accumulation of HA cable-like structures that are adhesive for leukocytes. Additionally, IBD mucosal endothelial cells produce higher levels of cell surface HA in response to TNF-alpha than non-IBD control cells. Therefore, HA deposition is an early event in inflamed gut tissue, preceding and likely promoting leukocyte infiltration.