An in vitro study was conducted to determine the effects of defaunation (removal of protozoa) and forage sources (rice straw, ryegrass and tall fescue) on ruminal fermentation characteristics, methane (CH4) production and degradation by rumen microbes. Sodium lauryl sulfate, as a defaunation reagent, was added into the mixed culture solution to remove ruminal protozoa at a concentration of 0.375 mg/ml. Pure cellulose (0.64 g, Sigma, C8002) and three forage sources were incubated in the bottle of culture solution of mixed rumen microbes (faunation) or defaunation for up to 24 h. The concentration of ammonia-N was high under condition of defaunation compared to that from faunation in all incubations (p<0.001). Total VFA concentration was increased at 3, 6 and 12 h (p<0.05~p<0.01) but was decreased at 24 h incubation (p<0.001) under condition of defaunation. Defaunation decreased acetate (p<0.001) and butyrate (p<0.001) proportions at 6, 12 and 24 h incubation times, but increased propionate (p<0.001) proportion at all incubation times for forages. Effective degradability of dry matter was decreased by defaunation (p<0.001). Defaunation not only decreased total gas (p<0.001) and CO2 (p<0.01~0.001) production at 12 and 24 h incubations, but reduced CH4 production (p<0.001) at all incubation times for all forages. The CH4 production, regardless of defaunation, in order of forage sources were rice straw > tall fescue > ryegrass > cellulose (p<0.001) up to 24 h incubation.