The objective of the present study was to analyze the effects of yeast-fermented rubber seed kernels (YERSEK) on the feed intake, hematology, microbial protein synthesis, milk yield, and milk composition in dairy cows. Six crossbred Holstein Friesian (HF) × Thai lactating dairy cows with 110 ± 10 days in milk were randomly assigned to three different amounts of YERSEK at 0%, 10%, and 20% in a concentrate mixture using a 3 × 3 repeated Latin square design. Cows were fed with concentrate diets at a concentrate-to-milk yield ratio of 1:1.5, with rice straw fed ad libitum. The inclusion of YERSEK did not adversely affect feed intake, nutrient intake, or digestibility (p > 0.05), whereas ether extract intake and digestibility linearly increased in dairy cows receiving YERSEK (p < 0.01). Increasing YERSEK levels did not adversely affect blood urea nitrogen (BUN) levels, hematological parameters, or microbial protein synthesis (p > 0.05). Supplementation of YERSEK did not influence milk production, lactose, or protein levels (p > 0.05). However, milk fat and total solids decreased linearly (p < 0.05) with the addition of YERSEK. In conclusion, in a concentrate diet, YERSEK could be used as a protein source without negative effects on feed intake, digestibility, hematology, microbial protein synthesis, or milk yield. However, it reduced the milk fat and total solids of tropical lactating dairy cows.