This study was conducted to evaluate the nutritional value of total mixed rations (TMR) containing rice grain in an in vitro rumen fermentation system. Three types of grains (corn, wheat, and rice), timothy, and soybean meal (SBM) were used to prepare the experimental TMR: Corn TMR, Wheat TMR, and Rice TMR. The rumen fermentation characteristics of all the experimental TMRs were evaluated by an in vitro anaerobic system using rumen fluid for 24 and 48 h. The digestibility of the nutrients (dry matter [DM], crude protein [CP], and neutral detergent fiber [NDF]), pH, ammonia (NH-N), and volatile fatty acids (VFA) were determined. Rice TMR showed a higher DM digestibility than that of the Corn TMR at 48 h (p < 0.05). In all treatments, the CP digestibility was more than 80% at 48 h, but no significant differences were observed among the treatments. The NDF digestibility tended to be the lowest in the Wheat TMR (p = 0.06), and the pH tended to be the lowest in the Rice TMR (p = 0.09) among the treatments for the 48 h incubation. The Wheat TMR had the highest NH-N concentration among the treatments (p < 0.01). Rice TMR had a lowest total VFA concentration among the treatments (p = 0.05) at 24 h, but no significant differences were observed at 48 h. Based on this in vitro result, it was considered that a rice grain has the potential to replace conventional grain ingredients when the TMR was formulated.