This study was conducted to investigate for the natural methane emission inhibitor as a feed additive no adversely effect on rumen fermentation. Five different Control (Wheat barn (0.05 g), MRA(Methane Reduction Additive)-1 (Allium fistulosum L. (0.05 g)), MRA-2 (Sodium Lauryl Sulfate (0.025 g) +Wheat barn (0.025 g) mixed), MRA-3 (Sodium Dodecyl Sulfate (0.025 g) + Wheat barn (0.025 g) mixed), and MRA-4 (Allium fistulosum L. (0.02 g) + Tannic acid (0.02 g) + Wheat barn (0.01 g) mixed) contents were used to perform 3, 6, 9, 12, 24 and 48 h incubation for in vitro fermentation. Ruminal pH values were ranged within normal ruminal microbial fermentation. Dry matter digestibility was not significantly different across the treatments during the whole fermentation time. Also, the result of microbial growth had no adversely effect on during the whole fermentation time. At 24 h, methane emission was significantly lower (P<0.05) than all treatments except to MRA-1. Especially, MRA-4 carbon dioxide emission was significantly lower (P<0.05) than control at 9, 24 and 48 h incubation. In addition MRA-4 propionate concentration was significantly higher (P<0.05) than control at 24 h incubation. The result of RT-PCR Ciliate-associated methanogens were significantly lower (P<0.05) at MRA-1, MRA-3 and MRA-4 than control at 24 h incubation. Based on the present results, MRA-4 could be suggestible methane emission inhibitor as a natural feed additive.