Abstract: Background: Studies on diet and high-density lipoprotein (HDL) subspecies distribution are limited. Objective: We examined the relationship between macronutrient composition and lipoprotein particle size and HDL subspecies independent of genetic effects by studying monozygotic (MZ) twins. Methods: 24 healthy MZ twin pairs aged 23–33 years were identified from two longitudinal population-based studies, FinnTwin16 and FinnTwin12. Total energy and nutrient intake were assessed with 3-day food records and physical activity was measured by the Baecke index. HDL subspecies distribution was determined by non-denaturing gradient gel electrophoresis. Associations between diet composition and HDL mean particle size were determined by multivariate nutrient density models adjusted for confounding variables. Results: Substituting one energy percentage from omega-3 polyunsaturated fatty acids (n-3 PUFAs) for a corresponding amount of energy from other type of fats was related to changes in the relative proportions of the HDL subspecies 2b, 3a and 3b toward a larger mean particle size in men (β ±SE: 1.00±0.26nm, p =0.004) and women (β ±SE: 0.90±0.21nm, p =0.001). This association remained significant in analyses controlling for genetic and shared environmental influences using within-pair differences of the measures in MZ twin pairs (β ±SE: 0.37±0.14nm, p =0.019). Twins with the higher n-3 PUFA intake had significantly higher proportions of large HDL2b particles and lower proportions of smaller-sized HDL3a and HDL3b particles as compared to their co-twins with lower intakes (p <0.05). Conclusions: Our data suggest that n-3 PUFA intake is associated with a favorable change in the distribution of HDL subspecies towards larger particles independent of genetic and shared environmental factors. [Copyright &y& Elsevier]