The objective of the present study was to examine the potential utilization of the yeast strain Pichia guilliermondii (Levica 27) on the ruminal fermentation of corn stover. Two in vitro treatments were evaluated: (A) filtered rumen liquid + powdered corn stover + yeast-free culture medium and (B) filtered rumen liquid + powdered corn stover + culture medium inoculated with yeast. Samples were collected at 0, 6, 12, and 24 h and examined for effects on culture pH, ammonia nitrogen (NH3-N), the fiber digestibility percentages of neutral detergent fiber and acid detergent fiber, the digestibility of dry matter, and the concentration of volatile fatty acids (VFAs). The results showed that the addition of Levica 27 had no effect on the ruminal pH, NH3-N or the digestibility indicators (p > 0.05). However, at 12 h, the inclusion of Levica 27 increased (p < 0.05) the molar concentrations of total VFAs and acetic and propionic acids in ruminal fluid and decreased (p < 0.05) the C2:C3 ratio. It is concluded that Levica 27 seems to have a stimulating effect on ruminal fermentation over a range within 6 and 12 h, a result that should be taken into account when in vivo studies are performed with this yeast strain.