Breast milk reduces the incidence of necrotizing enterocolitis (NEC). BH4 is a cofactor for endothelial NOS (eNOS). Reduced BH4 levels, or its oxidation to dihydrobiopterin (BH2), uncouple eNOS resulting in formation of reactive oxygen species (ROS) that have been implicated in the pathogenesis of NEC. We evaluated colostrum and mature breast milk, as well as infant formula, BH4 and BH2 content. In addition, we tested the BH4 effect on the newborn rat mesenteric arterial vascular tone. BH4 and BH2 content increased 3-fold in mature breast milk, when compared with colostrum (p < 0.01), without a change in their ratio. Infant formula had a negligible BH4 content and lower biopterins ratio, when compared with breast milk. eNOS is the predominant synthase isoform in newborn rat mesenteric arteries. In the presence of BH4, mesenteric arteries contracted less to thromboxane A₂ analog U46619 (p < 0.01) and this effect was abolished following eNOS inhibition. BH4 (10⁻⁶ M) vasorelaxed the newborn rat mesenteric arteries. We conclude that when compared with infant formula, breast milk has a high BH4 content that increases as breastfeeding continues. Given its mesenteric arterial vasorelaxing effect, BH4 may play an important role in the reduced NEC incidence among breast fed infants.