Abstract: This research was conducted to investigate the effects of exogenous fibrolytic enzyme on in vitro ruminal fermentation and microbial populations with substrates in different Forage to Concentrate ratios (F:C). Four levels (0, 40, 80 and 120 U g-1 dry matter substrate) of fibrolytic enzyme were supplemented to buffered rumen fluid and incubated with three different F:C ratios (80:20, 50:50 and 20:80) substrates at 39°C. After 24 h incubation, the results showed that in three different F:C ratios, fibrolytic enzyme significantly increased (p<0.001) total gas production, in vitro dry matter disappearance, concentration of total volatile fatty acid and molar proportion of propionate and decreased (p<0.001) pH but it did not significantly (p>0.05) affect molar proportion of butyrate. And in the high (F:C 80:20) and medium (F:C 50:50) forage substrates, fibrolytic enzyme increased (p<0.05) the molar proportion of propionate and decreased (p<0.05) the ratio of acetate to propionate but the effects did not observed in high concentrate (F:C 20:80) substrates. It was also found that fibrolytic enzyme increased (p<0.05) the number of Fibrobacter succinogenes and methanogens in the F:C 80:20 substrate but there was no significant (p>0.05) effects on microbial populations in the F:C 50:50 and 20:80 substrates. The results indicated that the fibrolytic enzyme improved the degradation of substrates and had different effects on the ruminal fermentation pattern and microbial populations under three F:C ratio substrates. The efficiency of fibrolytic enzyme on high and medium forage substrates was greater than low forage substrates.