In in vitro fermentation studies, feed samples can either be included in the in vitro rumen medium using filter bags or can be directly dispersed. The objective of this study was to investigate the effects of different pore sizes of filter bags on the rumen fermentation characteristics in an in vitro system. Corn, soybean meal, and timothy were ground to pass through a 1.0-mm screen and were formulated in the ratio of 70:7:23 based on DM, respectively. The formulated experimental diet (2g/DM) was put in F57 filter bags and R510 nylon bags (Ankom®) which pore sizes were 25 and 50 μm, respectively. An in vitro study was conducted to determine the rumen fermentation characteristics for 3, 6, 12, 24, and 48 h and rumen microbial community at 48 h of incubation. A significantly higher production of gas was observed in the R510 bags than in F57 at all the incubation times (p<0.01). IVDMD (p<0.01) and IVNDFD (p<0.01) were significantly higher, whereas pH (p<0.01) and NH3-N (p<0.01) were lower when R510 bags were used. In the VFA composition, acetate and butyrate were significantly higher (p<0.01) in R510 bags, and propionate and total VFA concentration did not differ (p=0.55 and 0.25, respectively) between F57 and R510 bags. The log copy numbers of bacteria and protozoa did not differ (p=0.69 and 0.94, respectively) between F57 and R510 bags, whereas those of fungi were significantly higher in R510 than in F57 bags (p<0.01). Therefore, the use of R510 may reflect actual rumen fermentation characteristics more precisely than those of F57 because increased gas production, nutrient digestibility and acetate, butyrate proportion were founded in R510.