Differences in vitamin and carotenoids content of human milk (HM) produced for infants born at term and preterm is poorly understood. In this study, HM was collected weekly for four and two months post-partum for preterm and term groups, respectively. Nutrients of interest, from single full breast expressions were measured by liquid chromatography coupled with mass spectrometry. Microbiological assay was employed for vitamin B ₁₂ . When compared at equivalent post-partum age, vitamins B ₁ , B ₂ , B ₆ , and B ₉ were significantly higher in preterm than in term HM, but only during the first two weeks. No significant differences were observed for A, E, B ₃ and B ₁₂ between groups. Lycopene was the only carotenoid exhibiting a significant higher concentration in term than in preterm HM between weeks 1 and 4 post-partum. When compared at equivalent post-menstrual age, preterm milk was significantly higher for vitamins B ₁ , B ₂ , B ₃ , B ₆ and B ₉ and lower levels of vitamins A, E, β-carotene, β-cryptoxanthin, lutein, zeaxanthin and lycopene compared to their term counterparts. These results suggest that preterm breastfed infants at term equivalent age may receive lower amounts of these micronutrients than breast-fed term neonates, possibly highlighting the need to supplement or fortify their nutritional intake with vitamins and carotenoids. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT #02052245.