Abstract Background Few studies in recent years have demonstrated the effect of maternal diet on fatty acid composition of human milk. Methods Fourteen free-living lactating women participated in a cross-over dietary intervention study, consuming a low fat diet (17.6% of energy as fat, 14.4% of energy as protein, 68.0% of energy as carbohydrate) and a high fat diet (40.3% of energy as fat, 14.4% of energy as protein, 45.3% of energy as carbohydrate) each for periods of 4 days, in randomised order. Each mother was her own control. Mature milk samples were collected during each period and analysed for medium and long chain fatty acids. Results The concentration of medium chain fatty acids (MCFA), was 13.6% in breast milk for the low fat diet compared to 11.4% for the high fat (p < 0.05). Arachidonic acid (C20:4n-6) levels were significantly higher in breast milk when women consumed the low fat diet. Increased dietary intake of stearic acid (C18:0) and alpha-linolenic acid (C18:3n-3) on the high fat diet significantly increased proportions of these fatty acids in breast milk (p < 0.05) in 4 days. Conclusions Changing maternal dietary fat intake has a rapid response in terms of changes to fatty acids in breast milk.