Abstract: This study was conducted to isolate rumen yeast and evaluate its possible role in changing fermentation characteristics by using in vitro fermentation with three different substrates. A yeast strain with a fast growth rate belonging to the Meyerozyma genus was isolated through screening, growth curve determination and identification of 26S rDNA using a selective medium of yeast composed of goat and dairy cow ruminal fluid. The effect of this isolated culture on ruminal fermentation was evaluated with three substrates: Chinese wildrye plus concentrate mixture, potato starch and corn starch. Ruminal fluid from dairy cattle was used as an inoculant. The results showed that with the Chinese wildrye plus concentrate mixture as substrate, yeast addition decreased ruminal pH and the concentration of lactic acid (P<0.001), while it increased the concentration of propionic acid and the digestibility of dry matter (P<0.05). The addition of yeast decreased ruminal pH and lactic concentration (P<0.001) in the corn starch substrate. Moreover, the addition of yeast culture decreased ruminal pH (P<0.05) and increased the concentration of propionic acid (P<0.05). However, total gas production and concentrations of ammonia-N, acetic acid, butyric acid, isobutyric acid, valeric acid and isovaleric acid were not affected (P>0.05) by yeast addition in the three substrate groups. The real-time PCR data showed that isolate addition increased the number of total bacteria in the Chinese wildrye plus concentrate and potato starch substrates. Results indicate that the rumen yeast isolates can improve the rumen’s dietary degradation capability and reduce lactic acid levels for the Chinese wildrye plus concentrate and corn starch substrates. This suggests that the yeast isolates could improve digestibility efficiency in ways that enhance the propionate production and the growth of ruminal bacteria.