The aim of this study was to evaluate the nutrient digestibility, ruminal fermentation and microbial protein synthesis of mid- lactating cows fed high dietary levels of monensin. Twelve Holstein cows were distributed into four 3 x 3 latin squares and assigned to the following treatments: control (CON), monensin 24 (M24, addition of 24 mg monensin/kg diet DM) and monensin 48 (M48, addition of 48 mg monensin/kg diet DM). Dietary levels of monensin linearly decreased dry matter intake without altering the nutrient total apparent digestibility. Monensin linearly increased ruminal fluid pH and acetate concentration. Monensin quadratically affected ruminal total short chain fatty acids, propionate concentration, acetate and propionate production and acetate to propionate ratio. Furthermore, monensin linearly increased the efficiency of microbial protein synthesis.