To observe the effect of smoking habit on age-related serum lipid levels, we examined a large cohort of Japanese cross-sectionally and longitudinally. The participants included 103,648 Japanese men and women 17-94 years of age, who had received annual health examinations from 1989 to 2003. In cross-sectional analysis, total and LDL cholesterol levels of smokers were lower than those of nonsmokers up to an elderly age in men and up to middle age in women. Smoking was associated with decreased HDL cholesterol levels up to the 65-74 years age group in men and 55-64 years in women. The triglyceride levels were higher in smokers in both genders than those of nonsmokers below 55-64 years. In the longitudinal analysis, although smoking was associated with lower total and LDL cholesterol up to 60 years of age in women, beyond the sixties an inverted association was observed. The associations of smoking with lower LDL cholesterol levels in men and lower HDL cholesterol in both genders were fairly consistent at any given age. The increase of triglyceride levels in female smokers remained rather constant between 25 and 75 years, whereas the increase in triglyceride levels in male smokers was greater with older ages up to middle age. These results suggest that the effect of smoking on the serum lipid levels is dependent on age and gender.