The disease process of ulcerative colitis (UC) is associated with a block in β-oxidation of short chain fatty acid in colonic epithelial cells which can be reproduced by exposure of cells to sulfides. The aim of the current work was to assess the level in the β-oxidation pathway at which sulfides might be inhibitory in human colonocytes. Isolated human colonocytes from cases without colitis (n = 12) were exposed to sulfide (1.5 mM) in the presence or absence of exogenous CoA and ATP. Short chain acyl-CoA esters were measured by a high performance liquid chromatographic assay. 14CO2 generation was measured from [1-14C]butyrate and [6-14C]glucose. 14CO2 from butyrate was significantly reduced (p < 0.001) by sulfide. When colonocytes were incubated with hydrogen sulfide in the presence of CoA and ATP, butyryl-CoA concentration was increased (p < 0.01), while crotonyl-CoA (p < 0.01) and acetyl-CoA (p < 0.01) concentrations were decreased. These results show that sulfides inhibit short chain acyl-CoA dehydrogenase. As oxidation of n-butyrate governs the epithelial barrier function of colonocytes the functional activity of short chain acyl-CoA dehydrogenase may be critical in maintaining colonic mucosal integrity. Maintaining the functional activity of dehydrogenases could be an important determinant in the expression of ulcerative colitis.