OBJECTIVE: This study assessed the impact of supplementing the diet of women during pregnancy and lactation with fish oil containing the omega-3 fatty acid docosahexaenoic acid, and its influence on the composition of human milk. METHODS: The sample comprised 60 women aged 18 to 38 years with appropriate dietary pattern, all of them healthy and nonsmokers. The intervention consisted of a daily supplementation with fish oil capsules that corresponded to a daily intake of 315mg of docosahexaenoic acid and 80mg of eicosapentaenoic acid during the third trimester of pregnancy and the first three months postpartum. The total fat content and fatty acid profile of their milk were determined by creamatocrit and gas chromatography. Descriptive statistics were used for data analysis and the significance level was set at p<0.05. RESULTS: There was no statistical difference between the fat contents of the study (fish oil capsules) and control (capsules containing corn starch as filler) groups. However, the milk of women taking fish oil contained higher docosahexaenoic and eicosapentaenoic acid levels 30 and 60 days after delivery. These results demonstrate that high omega-3 intake can influence its concentration in human milk. CONCLUSIONS: Given the importance of docosahexaenoic acid in the neonatal period, it is appropriate for pregnant and breastfeeding women to supplement on long-chain polyunsaturated fatty acids, which may be done by adding fish oil to the regular diet.