This experiment was conducted to study the effects of Peptide Nitrogen Source (PNS) infusion on ruminal fermentation pattern, nutrient digestibility, and microbial nitrogen production. Three ruminally cannulated Holstein dairy cows BW=682 kg and DIM=210d were assigned in a 3×3 Latin Square of 21-d periods. The first 14 d’s were considered as adaptation period and the last 7 d as sample collection period. Three levels of sodium caseinate as PNS were used in the experiment (0, 50 and 100gr/d considered as diets 1, 2 and 3 respectively). PNS was infused directly into rumen of the fistulated cows just before the offer of morning diet. Purine derivatives method was ebpolyed for daily microbial nitrogen synthesis estimation. Ruminal Ammonia Nitrogen (NH3-N) and Peptide Nitrogen (Pep-N) concentrations (P<0.01), as well as NH3-N to Pep-N ratio (0.58, 0.70 and 0.82 for diets 1, 2 and 3 respectively) were increased by infusion of PNS (P<0.05). Ruminal Volatile Fatty Acids (VFA) concentration bears a tendency to increase (P=0.07) but not significantly by infusion of casein. Acetate increased linearly (P<0.01) and there were no specific changes observed for propionate and butyrate through treatments. The Branched Chain Volatile Fatty Acids (BCVFAs) (i.e. valerate and isovalerate) increased significantly by PNS infusion. Ruminally PNS infusion had a significant effect on NDF, ADF (P<0.05) and especially hemicellulose (P<0.01) digestibility. Volume of urine was significantly affected significantly (P<0.01) by casein infusion in cannulated cows and urine creatinine, uric acid and allantion concentrations increased by PNS infusion. Daily microbial nitrogen yield estimated from purine derivatives (222.86, 238.75 and 257.51 g/d for diets 1, 2 and 3 respectively) were significantly increased through casein infusion into rumen (P<0.01). Fiber digestibility was improved, acetate and BCVFAs increased and microbial nitrogen yield increased by PNS, but a high level of PNS decreased nitrogen efficiency while increasing nitrogenous excretion and BUN. It could be concluded that ruminal microbes need some of their N requirement as peptide and the optimal fiber digestibility and nitrogen utilization efficiency and microbial nitrogen yield was gained when NH3-N/ Pep-N was 0.70 and lower than this value fiber digestibility and microbial nitrogen synthesis decrease and while higher than this value nitrogen utilization efficiency decreases.