Epidemiologic studies have revealed that the protective association of high-density lipoprotein cholesterol (HDL-C) with CHD is stronger in older men and younger women. We aimed to investigate sex differences in the relation of HDL-C to progression of carotid intima-media thickness (IMT) (an indicator of subclinical atherosclerosis) in middle age. IMT progression and serum HDL-C were determined for a cohort of 500 women and men aged 40-60 years over three examinations (1.5-year intervals). IMT at baseline was inversely associated with serum levels of HDL-C and the associations were comparable in women and men. However, in multivariate longitudinal growth models adjusting for potential confounders, IMT progression was inversely associated with serum levels of HDL-C in men, but directly associated in women (p=0.0007 for interaction). Our results suggest that although HDL-C was protective against progression of carotid atherosclerosis in middle-aged men, anti-atherogenic effects of HDL may diminish in women around the age of menopause.