Diets consumed in industrialized countries are rich in fat and increase the incidence of atherosclerosis, a process reported to be influenced by gender. Considering the anti-atherogenic role attributed to serum Paraoxonase 1 (PON1) activity, and given the pro-atherogenic effects described for saturated fatty acids (SFA), as opposed to the beneficial ones conferred to monounsaturated fatty acids (MUFA), the aim of this study was to investigate the response of male and female rat serum PON1 activity and its related factors to a high-fat (HF), hypercaloric diet (fat representing 55.2% of the energy) containing similar amounts of SFA and MUFA. The HF diet feeding did not alter total body weight, but increased adiposity. Nevertheless, and in spite of the increased adiposity, the HF diet did not entail a more pro-inflammatory serum adipokine or lipid profile or increased lipid peroxidation. Paraoxonase activity was reduced in both male and female HF fed rats, due to a reduction of PON1 mRNA levels in males and to a reduced stability and/or number of HDL particles responsible for PON1 transport in females. Both the maintenance of body weight and the MUFA content in the diet would be among the factors responsible for the attenuation of the negative effects usually related to excessive fat intake and for the reduction in PON activity, whose antioxidant activity would be less necessary in this situation.