This study was conducted to examine the effects of defaunation (removal of live protozoa) on fermentation characteristics, degradation of ryegrass hay and CH4 (methane) production by rumen microbes when incubated with plant oils (SO, sunflower oil and LO, linseed oil) in vitro. Sodium lauryl sulfate (0.000375 g/ml) as a defaunation reagent was added into the culture solution and incubated anaerobically up to 24 h at 39℃. pH from defaunation was increased for all treatments from 6 h incubation times (p<0.01-0.001) compared with those from fauantion. Concentration of ammonia-N from defaunation is higher than that from faunation at 3 h (p<0.001), 12 h (p<0.05) and 24 h (p<0.001) incubation times. Defaunation decreased (p<0.01-0.001) total volatile fatty acid concentration at all incubation times. Molar proportions of C2 (acetate, p<0.05-0.001) and butyrate (p<0.01-0.001) were also decreased by defaunation at all incubation times. Molar proportion of C3 (propionate), however, was increased by defaunation at all incubation times (p<0.001). Thus the rate of C2 to C3 was decreased by defaunation at all incubation times (p<0.001). Defaunation decreased ED (effective degradability) of dry matter (p<0.001) and ED of neutral detergent fiber (p<0.001) of ryegrass hay. Defaunation decreased total gas, CH4 production, CH4 % in total gas and CH4/CO2 at all incubation times (p<0.001). Oil supplementation decreased total gas (p<0.05-0.001), CH4 production (p<0.001) and CH4 % in total gas (p<0.001) compared with control at all incubation times. The result of this study showed that defaunation combined with oil supplementation may cause an alteration of microbial communities and further medicate the fermentation pattern, resulting in both reduction of degradation of ryegrass hay and CH4 production. No difference, however, was observed in all the examinations between SO and LO.