Rumen-protected conjugated linoleic acid (CLA) reportedly improves fertility in lactating dairy cows by reducing the postpartum interval to first ovulation and enhancing the circulating insulin-like growth factor-I (IGF-I) levels. The objectives of this study were to evaluate the blood metabolites, hormones, follicular fluid (FF) and liver for the effect of CLA supplementation (50 g top-dressed daily from 15 days pre-partum to 65 days in milk -DIM -). Pre-partum Holstein cows (n = 24) were assigned to two treatments: a Control and CLA group (n = 12 cows/group). Dry matter intake (DMI) and milk production were recorded daily. At 26 DIM, ovulation was synchronized and at 34 DIM, plasma and FF were analysed for paraoxonase 1(PON) levels. Moreover plasma was analysed for IGF binding protein 2 and 3 (IGFBP). From 34 DIM, blood samples and FF from follicles >9 mm were collected and analysed for estradiol, progesterone, IGF-I and lipoproteins. A liver biopsy was performed at 65 DIM and analysed for the expression of IGF-I, growth hormone receptor (GHR), pyruvate carboxylase (PC) and cytosolic phosphoenolpyruvate carboxykinase (PECK). CLA supplemented cows, compared to the control group, recorded a significant lower milk fat production, improved DMI and energy balance and recorded significant increased plasma concentrations of IGF-I, cholesterol, low density lipoprotein (LDL) and IGFBP-3 (interaction treatment x DIM). The concentration of IGF-I, high density lipoprotein (HDL) and LDL tended to be higher in FF than plasma. This study confirms the positive effects of dietary CLA supplementation on the metabolism, by improving the energy intake and reducing the negative energy balance. Moreover, the improvement of plasma IGF-I levels observed in this study, coupled with a better energy balance support previous studies showing a positive effect of CLA supplementation on reproduction. However, CLA did not alter the plasma and the FF concentration of PON, nor the liver gene expression.