This study investigated whether the interaction of protein level and grain type can affect milk production, nutrient digestibility and rumen fermentation in primiparous Holstein cows. Four dietary treatments were used: high-protein with barley as the only grain source, HP-B; (2) high-protein with an equal mix of barley and maize, HP-BM; (3) low-protein with barley as the only grain source, LP-B and (4) low-protein with equal proportions of barley and maize, LP-BM. High-protein diets showed no improvement in milk or protein yield compared with low-protein, but barley and maize mix diets increased energy-corrected milk yield and fat yield compared with barley-only diets. The highest total apparent digestibility of dry matter, organic matter and neutral detergent fibre was observed for LP-BM whereas HP-BM showed the greatest crude protein digestibility. Treatment had no effect on total volatile fatty acid concentrations, molar proportion of acetate and propionate and acetate to propionate ratio. The lowest ruminal pH was observed for LP-B. High-protein diets resulted in greater concentrations of ammonia nitrogen (N), urinary N, blood and milk urea nitrogen compared with low-protein diets, whereas low-protein diets showed better nitrogen utilization efficiency. This study showed that primiparous lactating cows do not benefit from high-protein diets with different fermentation rates of grain sources, but barley and maize diets may improve milk production performance, ruminal fermentation and pH under the present dietary conditions. The current results on milk production performance should be interpreted with caution due to the small number of cows used (eight in each treatment).