<i>Background/Aims:</i> Serum lipid concentrations worsen after the menopause because of estrogen deficiency, leading to an increased atherogenic pattern. It is known that serum paraoxonase (PON1) activity prevents the development of atherosclerosis. The aim of this cross-sectional study was to observe the effects of intranasal 17β-estradiol (300 µg/day) on serum PON1 and lipid levels in healthy postmenopausal women. <i>Methods:</i> 48 healthy, postmenopausal women were enrolled into this cross-sectional study. 28 subjects without an intact uterus and ovaries were using single-dose (300 µg/day) intranasal 17β-estradiol and 20 subjects with spontaneous natural menopause were not on any hormone therapy. Body mass index (BMI), blood pressure, serum follicle-stimulating hormone, estradiol, fasting glucose, insulin, lipid fractions and PON1 levels were measured. Homeostasis model assessment (HOMA-R) was used to estimate insulin resistance. <i>Results:</i> The higher estradiol, high-density lipoprotein and salt-stimulated paraoxonase (SSP) levels were observed in intranasal 17β-estradiol users in comparison with non-users. There were no statistically significant differences in BMI, blood pressures, other lipid fractions, basal paraoxonase, arylesterase, fasting glucose and insulin levels, HOMA-R between the groups. SSP was inversely associated with fasting insulin levels and HOMA-R. <i>Conclusion:</i> These observations may suggest that intranasal 17β-estradiol does not have harmful effects on the PON1 activity and lipid metabolism.