An in vitro experiment was conducted to examine the effects of defaunation (removal of protozoa) on ruminal fermentation characteristics, $CH_4$ production and degradation by rumen microbes when incubated with cereal grains (corn, wheat and rye). Sodium lauryl sulfate as a defaunation reagent was added into the culture solution at a concentration of 0.000375 g/ml, and incubated anaerobically for up to 12 h at $39^{circ}C$. Following defaunation, live protozoa in the culture solution were rarely observed by microscopic examination. A difference in pH was found among grains regardless of defaunation at all incubation times (p<0.01 to 0.001). Defaunation significantly decreased pH at 12 h (p<0.05) when rumen fluid was incubated with grains. Ammonia-N concentration was increased by defaunation for all grains at 6 h (p<0.05) and 12 h (p<0.05) incubation times. Total VFA concentration was increased by defaunation at 6 h (p<0.05) and 12 h (p<0.01) for all grains. Meanwhile, defaunation decreased acetate and butyrate proportions at 6 h (p<0.05, p<0.01) and 12 h (p<0.01, p<0.001), but increased the propionate proportion at 3 h, 6 h and 12 h incubation (p<0.01 to 0.001) for all grains. Defaunation increased in vitro effective degradability of DM (p<0.05). Production of total gas and $CO_2$ was decreased by defaunation for all grains at 1 h (p<0.05, p<0.05) and then increased at 6 h (p<0.05, p<0.05) and 12 h (p<0.05, p<0.05). $CH_4$ production was higher from faunation than from defaunation at all incubation times (p<0.05).