Aims/hypothesis: Dietary fatty acids may affect insulin sensitivity. Adipose tissue fatty acid composition partly reflects long-term dietary intake, but data from large studies regarding relationships with insulin sensitivity are lacking. We aimed to determine the association between adipose tissue fatty acids and insulin sensitivity in elderly Swedish men. Methods In a cross-sectional analysis of the community-based Uppsala Longitudinal Study of Adult Men (n?=?795, mean age 71 years), adipose tissue biopsies were obtained and fatty acid composition was determined by gas–liquid chromatography. Insulin sensitivity was measured directly by a euglycaemic clamp. Results Palmitic acid (16:0), the major saturated fatty acid (SFA) in the diet and in adipose tissue, was negatively correlated with insulin sensitivity (r?=?-0.14), as were 16:1 n-7 (r?=?-0.15), 20:3 n-6 (r?=?-0.31), 20:4 n-6 (r?=?-0.38), 22:4 n-6 (r?=?-0.37) and 22:5 n-3 (r?=?-0.24; p?<?0.001 for all). Some minor SFAs were positively correlated; 12:0 (r?=?0.46), 14:0 (r?=?0.32), 17:0 (r?=?0.21) and 18:0 (r?=?0.41; p?<?0.001 for all), as were essential polyunsaturated fatty acids (PUFAs) 18:2 n-6 (r?=?0.10, p?<?0.01) and 18:3 n-3 (r?=? 0.16, p?<?0.001). Docosahexaenoic acid (22:6 n-3) was negatively correlated (r?=?-0.11, p?<?0.01), whereas eicosapentaenoic acid (20:5 n-3) was not (r?=?-0.02, NS). Most associations diminished or disappeared in lean individuals, indicating an effect of obesity. Conclusions/interpretation Adipose tissue enriched with palmitic acid and depleted of essential PUFAs is associated with insulin resistance. The positive association between minor SFAs and insulin sensitivity merits further investigation.