Abstract A replicated 3×3 Latin square study was conducted to evaluate the effects of replacing a portion of ground corn in the diet with dietary glycerol on rumen environment, blood metabolites, and nutrient digestibility. Six rumen-cannulated Holstein cows, averaging 56±18 DIM and 38.0±8.2kg of milk/d, were used in the study. Experimental periods included 3wk for treatment adjustment period followed by 1wk for data collection. Diets were corn silage based and balanced to be isocaloric and isonitrogenous. Treatments were 0g of glycerol/d (control, CON), 200g of glycerol/d (G2), and 400g of glycerol/d (G4). Dry matter intake (DMI) decreased as the amount of glycerol fed increased. Milk yield was higher for CON and G2 cows in comparison with G4 cows. Milk fat percentage and yield were reduced when glycerol was fed compared with CON cows but increased the milk protein percentage at the highest concentration of dietary glycerol. These changes resulted in decreased energy-corrected milk yield and efficiency (milk/DMI) in diets supplemented with G4 compared with CON. No differences were observed in ruminal pH and ammonia concentrations. Molar proportions of acetate, valerate, and acetate:propionate ratio decreased, whereas propionate increased as the amount glycerol fed increased. Molar proportions of butyrate were greatest when glycerol was included in the diet compared with CON. Nutrient intake and digestion were not different among treatments. Results of this trial suggest that feeding increasing amounts of glycerol may decrease DMI and alters ruminal fermentation, resulting in reduced yield of milk, fat, and energy-corrected milk.