The objective of this study was to investigate the effects of disodium fumarate (FUM; 3.75 mmol/d) on the fermentation of a 60:40 grass hay:concentrate substrate in Rusitec fermenters. Each of eight fermenters received daily 20 g of substrate dry matter (DM). FUM treatment tended to increase (P=0.076) substrate DM disappearance after 6 h of incubation (39.8 vs 42.2%), but no effects were detected after 48 h (P=0.830). Daily production of acetate, propionate and total volatile fatty acids (VFA) was increased by 11, 23 and 11%, respectively, in FUM fermenters, but methane production was not affected (P=0.167). FUM treatment decreased (P<0.001) the acetate:propionate (2.92 vs 2.62) and methane:VFA (0.248 vs 0.208) ratios and increased the daily flow of microbial N (P=0.007; 140 vs 149 mg/d). In contrast, FUM did not affect (P=0.260 to 0.960) either the numbers of total and cellulolytic bacteria or the fibrolytic activity of ruminal fluid.