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The objectives in this study were to measure particle size and evaluate the effects of reducing alfalfa hay particle size on Holestein lactating dairy cows based on measurements through Penn State Particle Separator (PSPS). Twelve Holestein lactating dairy cows averaging 84±19 day in milk (DIM) and 580.1±55.1 kg body weight (BW) were assigned to a balanced change – over design along with four diets, three periods, three blocks and four cows per block. During each of the 28d period, animals were individually offered one of the four diets, which were identical in energy, protein and chemical composition, but differed only in particle size of alfalfa hay (Alfalfa hay was chopped using 10, 20, 40, and 100 mm screens). Distribution of particle size of alfalfa hay and total mixed rations (TMR), was determined through PSPS and it was observed that the amount of particles > 19.00 mm significantly decreased with decreasing alfalfa hay particle size. The geometric mean length of alfalfa hay particle size was 2.86, 4.44, 4.87, 7.36 mm for 10, 20, 40 and 100 mm screens, respectively. The geometric mean length of particle size in diets were 4.14, 5.15, 6.38 and 8.34mm for total mixed rations that included alfalfa hay chopped with 10, 20, 40 and 100 mm screens, respectively. Reducing alfalfa hay particle size increased dry matter intake linearly (22.08, 21.49, 21.04, 20.62 kg/ day for rations that included alfalfa hay chopped with 10, 20, 40 and 100 mm screens, respectively). Milk fat corrected (MFC, 4%) and fat percent were significantly (P<0.05) different in treatments: 27.86±3.29, 30.42±2.193, 32.45±5.15 and 35.88±5.92 kg/d; and 2.77±0.32, 3.43±0.40, 3.61±0.30 and 3.60±0.32 distributed alfalfa hay with Xgm of rations 1, 2, 3 and 4, respectively. Protein, lactose, and non fat total solids were affected by diets. Feed conversion ratios (dry matter in kg per kg milk) for rations 1, 2, 3 and 4 were 0.79, 0.71, 0.65 and 0.61, respectively. A significant (P<0.05) effect was observed for mean rumen pH, 5.58, 6.02, 6.38 and 6.37 for diets that contained chopped alfalfa with 10, 20, 40 and 100 mm screen, respectively, the difference being statistically significant. The treatments did not show any effect on blood metabolites.