Background: It is shown that kiwifruit elevates serum high-density lipoprotein cholesterol (HDL-C) levels and exhibits beneficial effects on human health due to its antioxidant potential. Objectives: This study aimed to investigate the impact of kiwifruit on the activity of the paraoxonase 1 (PON1) enzyme, as a main antioxidant enzyme in HDL functionality, in a high-fat diet (HFD). Methods: To this end, 42 male Syrian hamsters were divided into 6 groups including hamsters receiving a normal diet (the control normal group), a regular diet supplemented with kiwifruit at two concentrations (i.e., 1.86 g/kg and 3.73 g/kg), a HFD comprised of 15% butterfat + 0.05% cholesterol (the control high-fat group), and a HFD supplemented with kiwifruit at two concentrations (i.e., 1.86 and 3.73 g/kg) for 8 weeks. Results: The results showed that supplementation of kiwifruit to the HFD increased the levels of HDL-C and remarkably reduced the concentrations of oxidized low-density lipoprotein (ox-LDL) and malondialdehyde (MDA) compared with the control-HF group. In addition, the paraoxonase activity of PON1 significantly increased in HFD supplemented with kiwifruit (1.86 g/kg), and finally, arylesterase (ARE) activity increased in all treated groups when compared with untreated groups. Conclusion: Our findings suggested that kiwifruit can improve the lipid profile and prevent oxidative stress-induced by lipid peroxidation in hamsters receiving HFD, thus increasing the ARE and paraoxonase activities of PON1.