Retinol, α-tocopherol, and γ-tocopherol are fat-soluble vitamins acting as antioxidants via the prevention of lipid oxidation. Little is known about circulatory levels in healthy individuals. The present cross-sectional study aimed at elucidating the relationship between these antioxidants and clinical biomarkers in 206 male (median age 41 years, range 23-67) employees from companies located in the Okayama Prefecture, Japan. Subjects younger than 40 years (n = 94) showed a positive association of the frequency of alcohol consumption with the circulating retinol (β = 0.344, p = 0.001) and γ-tocopherol levels (β = 0.219, p = 0.041), and an inverse association of fast insulin with serum retinol (β = -0.301, p = 0.009). In participants older than 40 years (n = 112) we found that an inverse association of HOMA-R with serum retinol (β = -0.262, p = 0.021), α-tocopherol (β = -0.236, p = 0.035), and γ-tocopherol levels (β = -0.224, p = 0.052); and cigarette smoking was inversely associated with the levels of serum α-tocopherol (β = -0.286, p = 0.008) and γ-tocopherol (β = -0.229, p = 0.040). We further found negative relationships between serum ferritin and the retinol (β = -0.211, p = 0.032) and α-tocopherol levels (β = -0.223, p = 0.022) in men over 40 years of age. The present study suggests that the circulatory levels of antioxidant vitamins may modulate the action of insulin and that higher levels of iron might decrease the levels of antioxidant vitamins in the blood.