This study was conducted to evaluate changes to in vitro ruminal fluid fermentation due to the use of corn-derived dried distillers' grains with solubles (corn DDGS) as a partial or complete replacement for crushed cereal and oilseed meals in the fermentation substrate. The control substrate consisted of mixed cereal and oilseed meals (barley, wheat, soybean and rapeseed), while the experimental substrates were the same meals with increasing portions replaced with corn DDGS. Including corn DDGS decreased the total VFA concentration (P<0.05), ammonia level (P<0.001), methane emission (P<0.05) and total gas production (P<0.001) during microbial fermentation. Using DDGS-containing substrates did not change the proportions of acetate, propionate and butyrate, but did decrease the proportions of isobutyrate and isovalerate (P<0.001). The fermentation efficiency, VFA utilization index, cell yield coefficient and pH of the ruminal fluid also remained unchanged. The partial replacement of cereal and oilseed meals with corn DDGS had no deleterious effects on ruminal fluid fermentation.