Background/Aims: Because the neutral protease urokinase is important in control of cell adhesion and migration, the effects of the physiologically relevant fermentation product butyrate on urokinase secretion by colonic epithelium were examined. Methods: Secreted and cell-associated levels of urokinase and plasminogen activator inhibitor 1 were measured in colonic crypt cells within 24 hours of isolation from macroscopically normal mucosa of normal or cancer-bearing colons. Results: Butyrate caused a concentration-dependent inhibition of both secreted (56% ± 4% inhibition after 24-hour exposure to 1 mmol/L butyrate; n = 20; mean ± SEM; P < 0.001) and cell-associated urokinase content (35% ± 6%; P = 0.003). Acetate and propionate had minimal effects. Butyrate also stimulated plasminogen activator inhibitor 1 secretion by 25% ± 7% (P = 0.013). Net urokinase activities were suppressed in supernates and cell homogenates by butyrate. Levels of transcripts for urokinase and the inhibitor changed with butyrate exposure in parallel to the levels of secretion of the respective proteins. Cells from the cancer group showed significantly reduced inhibitor secretion and abnormal responses to butyrate (greater inhibition of urokinase secretion and no stimulation of inhibitor secretion), probably reflecting the diffuse disturbance of colonic epithelial biology associated with colorectal cancer. Conclusions: Butyrate has dual effects in markedly reducing colonic epithelial urokinase activity, and these may have important implications to understanding colonic epithelial physiology and the pathogenesis and treatment of colonic diseases.