The aim of this study was to evaluate the effect of total replacement of raw whole soybean (RAW) for roasted whole soybean (ROS) on the production performance of Holstein cows. Two experiments were carried out using a simple reversal design where RAW has been completely replaced by ROS. In experiment 1, 22 cows (175 +/- 60 days in milk) were used, and the dietary inclusion level of RAW or ROS was 3.7% of dry matter (DM). In experiment 2, 16 cows (130 +/- 50 days in milk) were used, and the dietary inclusion level of RAW or ROS was 11% of DM. In both experiments, ROS increased milk production by 1.1kgday(-1) without changing fat and protein production. Dry matter intake or milk urea nitrogen were not affected by dietary soy source. In experiment 2, plasma glucose concentration was decreased, and allantoin/creatinine ratio in urine tended to decrease in ROS. Experiment 2 also evaluated the nutrient digestibility and ruminal degradation kinetics of crude protein in two soybean sources. Roasting had no effect on the digestibility of DM, organic matter, and neutral detergent fiber. Roasted whole soybean had greater fraction B and lower protein degradation rate than did RAW; this showed that heat treatment was effective in increasing the rumen undegradable amino acid flow to the animal, which suggested a potential mechanism of action for improved performance observed in ROS.