The objective of this study is to determine whether a yeast culture of Saccharomyces cerevisiae has an effect on parameters of ruminal fermentation and whether the potential positive effects of yeast culture are associated with the yeast’s viability. For this purpose, live and autoclaved yeast cultures were tested simultaneously in the rumen simulation technique (Rusitec). Each fermentation vessel received daily 9 g of a basal diet consisting 5 g meadow hay and 4 g pelleted concentrate. Yeast preparations (1.5 g/day) were added with the feed. Compared to the control vessels, the pH of ruminal fluid was significantly (P<0.05) lower in the vessels supplemented with autoclaved yeast culture. Live yeast culture had no effect on the pH. Both yeast preparations resulted in a significant increase (P<0.05) in NH₃-N concentration, total and individual short-chain fatty acid (SCFA) production except valerate. The increase in NH₃-N concentration and propionate production was significantly higher (P<0.05) in the vessels supplemented with live yeast culture than that with autoclaved yeast culture. Also, the ratio of acetate to propionate was significantly lower (P<0.05) by live yeast culture, when compared with the control vessels. Organic matter digestibility was not significantly affected by both yeast preparations. In conclusion, these results indicated that the addition of live and autoclaved yeast cultures stimulated ruminal fermentation but this effect was more pronounced with live yeast culture.