Human milk fat is specially tailored to supply the developing infant with adequate and balanced nutrients. The present study aimed to quantify the composition of fatty acids, tocopherols and carotenoids in human milk, with special emphasis on the lactational changes. Colostrum, transitional and mature milk samples were collected longitudinally from the same forty-two healthy, well-nourished Chinese mothers. Fatty acids were quantified by GC with carotenoids (carotenes and xanthophylls) and tocopherols (α-, γ-tocopherol) determined by HPLC. Total fatty acid (TFA) content increased from 15·09 g/l in colostrum to 32·57 g/l in mature milk with the percentages of DHA and arachidonic acid (ARA) decreased. The ratio of n-6:n-3 PUFA and ARA:DHA remained constant during lactation at about 11:1 and 1·3:1, respectively. Both α-tocopherol and γ-tocopherol decreased over lactation with the ratio of α-:γ-tocopherol declined significantly from 7·21:1 to 4·21:1 (P < 0·001). Carotenoids all dropped from colostrum to mature milk as the less polar carotenes dropped by 88·67 %, while xanthophylls only dropped by 35·92 %. Lutein was predominated in both transitional and mature milk carotenoids (51·64–52·49 %), while colostrum carotenoids were mainly composed of lycopene (32·83 %) and β-carotene (30·78 %). The concentrations of tocopherols and xanthophylls but not carotenes were positively associated with TFA content in milk. These results suggested that colostrum and mature milk contained divergent lipid profiles and selective transfer mechanisms related to polarity might be involved. The present outcomes provide new insights for future breast-feeding studies, which also add in scientific evidences for the design of both initial and follow-on infant formulas.