This study was conducted to evaluate Raphanus sativus extracts to methane reduction in rumen. Five different levels of R. sativus extracts were used to investigate the most effective dosing level for the decrease of methane production in the rumen. The rumen fluid was collected from a cannulated one Hanwoo cow (BW=450±30 kg) consuming 600 g/kg timothy and 400 g/kg concentrate. On fermentation day, rumen fluid was collected at 2 hr postfeeding R. sativus extracts was dosed to achieve final concentration of 0, 1, 3, 5, 7, and 9% respectively, to fermentation bottles containing the mixture of rumen fluid and McDougall’s buffer and 300 mg of timothy was added as a substrate. The fermentation was conducted for 3, 6, 9, 12, 24, 48 and 72 hr incubation time at 39℃ with shaking. In vitro ruminal pH values were measured normal range for ruminal fermentation. Dry matter disappearance was significantly higher (p<0.05) at 3 hr incubation time 1, 3 and 5% doses than that of control. The highest methane reduction was observed in 12 hr incubation time 5, 7 and 9%. The carbon dioxide emission was also significantly (p<0.05) lower than that of control at 12 hr incubation time 5, 7 and 9%. The total volatile fatty acid was no significant difference between control and all doses level at 12 and 24 hr incubation time. At 24 hr incubation time, the result of real-time PCR were indicated that M. archea was significantly lower (p<0.05) at all doses level comparing to that of control. In conclusion, R. sativus extracts were significantly decreased methane emission. R. sativus extracts were significantly lower (p<0.05) than that of control at 12 hr incubation time 5, 7 and 9% and no adversely effect in rumen pH, dry matter disappearance and total VFA.