Increasing evidence suggests that fetal and neonatal nutrition impacts later health. The aims of the present study were to determine the effect of maternal dietary fat composition on intestinal phospholipid fatty acids and responsiveness to experimental colitis in suckling rat pups. Female rats were fed isocaloric diets varying only in fat composition throughout gestation and lactation. The oils used were high in n-3 (canola oil, 8% 18:3n-3), n-6 (safflower oil, 72% 18:2n-6), or n-9 (high oleic acid safflower oil, 78% 18:1n-9) fatty acids, n=6/ group. Colitis was induced on postnatal day 15 by intra-rectal dinitrobenzene sulfonic acid (DNBS) administration, with vehicle (50% ethanol) and procedure (0.9% saline) controls. Jejunal and colonic phospholipids and milk fatty acids were determined. The distal colon was assessed for macroscopic damage, histology and myeloperoxidase activity. The 18:2n-6 maternal diet increased n-6 fatty acids, whereas the 18:3n-3 diet increased n-3 fatty acids in milk and pup jejunal and colonic phospholipids. Maternal diet, milk and pup intestinal n-6/n-3 fatty acid ratios increased significantly in order: high 18:3n-3 < high 18:1n-9 < high 18:2n-6. DNBS administration in pups in the high 18:2n-6 group led to severe colitis with higher colonic damage scores and MPO activity than in 18:1n-9 and 18:3n-3 groups. High maternal dietary 18:3n-3 intake was associated with colonic damage scores and MPO activity that were not significantly different from ethanol controls. We demonstrate that maternal dietary fat influences the composition of intestinal lipids and responsiveness to experimental colitis in nursing offspring.