The aim of this study was to determine the effect of replacement of soybean meal (SBM, solvent extracted meal, 40% CP) with different levels of sesame meal (SSM, mechanically processing meal, 43.91%) on performance, milk composition, blood metabolites and profile of milk fatty acids in lactating dairy cows. Thus, eight Holstein dairy cattle were used in a replicated 4 x 4 latin square design during four 28-days periods. Treatments were control (no SSM supplementation) or replacement of 50, 75 or 100% of SBM with SSM. Cows were fed a total mix ration (TMR). The results showed that dry matter intake (DMI), total solid (TS), fat to protein ratio (FPR), milk protein yield (MP), milk lactose yield, and blood cholesterol, glucose and calcium concentrations were not affected (P>0.05) by experimental diets. However, milk yield average, fat corrected milk (FCM), feed efficiency (FE), milk fat (MF), solids non-fat (SNF) content, milk urea nitrogen (MUN), blood urea nitrogen (BUN) and triglyceride (TG) concentrations differed (P<0.05) among treatments. The concentration of cis-C18:1 in the milk of cows fed SSM diets were numerically greater compared with cows fed the control diet. Polyunsaturated fatty acids (PUFA) and total unsaturated fatty acids in milk of cows fed SSM diets were more than the control diet. Concentrations of C16:0 and saturated fatty acids in MF in the control diet were greater than those achieved for the SSM diets. Regardless of the SSM/SBM ratio, cows fed SSM had more unsaturated fatty acids in milk. Replacing SBM with SSM decreased (P<0.05) concentration of medium (MCFA) and long chain saturated fatty acids (SFA) and increased (P<0.05) long-chain polyunsaturated fatty acid (PUFA). Generally, SSM improved milk fatty acids profile, as a health index of human nutrition, but regarding the rate of milk production, 75% substitution of soybean with sesame is appropriate.