To evaluate effects of rumen protected methionie (RPM) on dairy cow performance, cow’s milk fatty acids profile and ruminal biohydrogenation of fatty acids Eight Multiparous Iranian Holstein cows with average 129± 19 days in milk (DIM) were used in a replicated (n= 4) latin square design with two periods of two weeks. Cows were fed with control diet without methionine supplement or a diet supplemented with 15g/day RPM (Mepron M ® ) per cow. Performance outcome was measured at each the end of each week and samples from milk and rumen fluid were collected weekly. Samples from cow’s rumen fluid and milk from wk-1 and wk-2 were mixed together and analysed for fatty acids profile as an overall mean. Milk samples were analyzed for fatty acids composition. Rumen fluid samples were also analyzed for the concentration of rumen biohydrogenation intermediates . Data on cow’s performance were analyzed for each wk separately and as overall means for each period. Dietary methionine supplementation did not affect cows dry matter intake through wk-1 and wk-2 but caused an increase (P=0.0434) in overall dry matter intake by methionine supplementation cows. Dry matter intake in wk-1 (P=0.1401) and wk-2 (P= 0.1068) tended to increase. RPM did not affect milk production, milk fat content and yield as well as lactose content or yield. Milk production in wk-2 (P= 0.0781) and overall (P= 0.1089) tended to increase by RPM diet. Milk fat content tended to increase as overall mean (P= 0.1089) while milk fat production tended to increase at wk-2 (P= 0.1062) and overall (P=0.0529); however, RPM increased protein content significantly through wk-1(P= 0.025), wk-2 (P= 0.0247) and overall period (P= 0.0039) and also milk protein yield at wk-2 (P= 0.0319) and overall experimental period (P= 0.0198). Lactose content at wk-1 (P= 0.1316), overall mean (P= 0.0529) and lactose production at wk-2 (0.0797) and also as overall mean (0.0979) tended to increase for diet supplemented with RPM. Energy corrected milk was increased significantly in wk-2 (P= 0.0360) and overall period (P= 0.0443) for diet containing RPM. Fat corrected milk (FCM) did not affect by RPM; however, FCM tended to increase in wk-1 (P=0.0598), wk-2 (P=0.0616) and overall trail period (P= 0.0602). Milk short chain fatty acids in RPM-supplemented cows were increased (P= 0.038) but medium and long chain fatty acids were not affected by RPM supplementation diet. Among individual short chain fatty acids, caprylic (P= 0.0295) and capric (P= 0.0299) acids were increased significantly by supplemented methionine diet. Butyric (P= 0.1492) and caproic (P= 0.0828) acids, tended to increase when, RPM was added to the diet. RPM caused significant increases in Stearic (P= 0.0466) and linolenic acid (P= 0.0378) acid content of ruminal fluid but trans 10, cis 12 conjugated linoleic acid (P= 0.0220) was reduced. Interestingly Other c:18 fatty acids were not altered significantly in rumen fluid. The present findings, indicate, that dietary supplementation of RPM can increase milk protein content and milk short chains fatty acids and also may alter rumen biohydrogenation process in dairy cows. Keywords: Rumen Protected Methionine, Milk Production, Milk Fat, Milk Fatty Acids Composition, Rumen Biohydrogenation