The objective of the study was to determine the effects of conjugated linoleic acid (CLA) supplement on milk yield and composition, blood metabolites and reproductive parameters in lactating Holstein dairy cows. Twenty Holstein dairy cows were randomly assigned to one of two dietary treatments: 1) supplementing 110 g per day of fat (control), 2) supplementing 120 g per day of rumen-protected CLA. The diets were formulated to be nutritionally isocaloric and isonitrogenous. The experimental period started 21 days pre-calving and continued until 60 days in milk (DIM). Treatments had no effect on dry matter intake (DMI), body weight (BW) and body condition score (BCS). The CLA treatment increased milk yield (3.04 kg per day and milk lactose concentration, but decreased milk fat concentration and, short and medium chain fatty acids concentrations. No treatment differences were observed in milk protein concentration, milk energy output and net energy balance. Serum concentrations of glucose, cholesterol, triglyceride (TG), insulin, insulin-like growth factor-1(IGF-1), estradiol and progesterone were higher in CLA treated cows when compared to cows fed on the control diet. Serum beta-hydroxybutyric acid (BHBA) concentration was reduced in cows fed on the CLA treatment. Days to first insemination and days open were not different between the two treatment groups. Cows fed on the CLA supplement had increased conception rate from the first service. The results indicated that cows fed on diets supplemented with CLA produced milk with decreased milk fat concentration whereas some related cow blood serum metabolic parameters associated with reproductive response were increased and resulted in an increased conception rate from the first service.