Arterial stiffness may be influenced by sex steroids and insulin; the association with fasting insulin level may be stronger in women than in men. Therefore, we analyzed the effects of sex steroid administration on (1) arterial stiffness and (2) the relationship between fasting insulin level and arterial stiffness. Twelve male-to-female transsexuals were treated with ethinyl estradiol and cyproterone acetate, and 18 female-to-male transsexuals were treated with testosterone esters, with assessments made at baseline and after 4 and 12 months. Changes in distensibility and compliance coefficients (DC and CC, respectively) of the common carotid artery, femoral artery (FA), and brachial artery (BA) were analyzed in relation to changes in fasting plasma levels of glucose, insulin, HDL-cholesterol, and triglycerides. After 4 months of estrogens and antiandrogens in men, significant reductions in the CC and DC of the FA (P=0.006 and P=0.04, respectively) and BA (P=0.04 and P=0.04, respectively) were observed. In women, testosterone, on average, did not affect DC or CC, but the changes in fasting insulin level were strongly negatively associated with changes in the CC and DC, especially in the FA and BA. These associations were significantly less strong in genetic men and were independent of age, mean arterial pressure, and glucose and lipid levels. This experimental study shows (1) that short-term administration of estrogens and antiandrogens increases FA and BA stiffness in men and (2) that the fasting insulin level is a stronger determinant of arterial stiffness in women than in men.