Paraoxonase (PON) possesses antiatherogenic potentials, but the distinct functions of PON members in alleviating atherosclerosis are not yet clear. This study aimed to evaluate the protective effects of hPON1 and hPON3 against atherosclerosis, and thereby exploring their synergistic mechanism in atherosclerosis development. We generated the recombinant adenovirus AdPON1 and AdPON3, which were capable of expressing hPON1 and hPON3. After AdPON1 and AdPON3 were injected intravenously into 5-week-old apolipoprotein E knockout mice, abundant hPON1 and hPON3 mRNA expression levels were detected. However, increase in serum lactonase activity was detected only in AdPON1-treated mice. Serum antioxidation and anti-inflammation capabilities in AdPON1-treated mice, reflected by malondialdehyde, total antioxidant capability and tumor necrosis factor-alpha levels, were greatly enhanced, whereas those in AdPON3-treated mice were not significantly affected. Nevertheless, histological analysis revealed that adenovirus-mediated expression of hPON1, hPON3 or both of them reduced atherosclerotic plaque area to a similar extent. Although no synergistic mechanism was detected in reducing arterial lesion size, hPON1 and hPON3 showed synergistic effects on promoting macrophage cholesterol efflux. In conclusion, hPON1 and hPON3 exhibited similar potentials in reducing arterial lesion size, but they exerted antiatherogenic effects in distinct ways.