<b><i>Background: </i></b>Based on animal studies, n-3 polyunsaturated fatty acids (PUFAs) have been suggested to lower the risk of obesity and inflammation. We aimed to investigate if, among humans, intake of n-3 PUFAs was associated with i) total body fat, ii) body fat distribution and iii) obesity-related inflammatory markers. <b><i>Methods:</i></b> The study population consisted of 1,212 healthy individuals with information on habitual food intake from food frequency questionnaires, six different measures of body fat, and levels of six circulating inflammatory markers. Multiple linear regression analysis of intakes of PUFAs in relation to outcomes were performed and adjusted for potential confounders. <b><i>Results: </i></b>Absolute n-3 PUFA intake, but not n-3/n-6, was inversely associated with the different measures of body fat. Among n-3 PUFA derivatives, only α-linolenic acid (ALA) was inversely associated with body fat measures. No significant interactions with the dietary macronutrient composition were observed. Pro-inflammatory cytokines were not associated with absolute PUFA intake, but the macrophage inflammatory protein-1α (MIP-1α) was associated with the n-3/n-6 ratio. <b><i>Conclusion:</i></b> In humans, intake of n-3 PUFAs, in particular ALA, is beneficially associated with body fatness. The favourable association is, however, not reflected in systemic levels of pro-inflammatory cytokines, nor is it influenced by macronutrients in the diet.