Background Maternal breast milk (MBM) is enriched in microRNAs, factors that regulate protein translation throughout the human body. MBM from mothers of term and preterm infants differ in nutrient, hormone, and bioactive factor composition, but the microRNA differences between these groups have not been compared. We hypothesized that gestational age at delivery influences microRNA in MBM, particularly microRNAs involved in immunologic and metabolic regulation. Methods MBM from mothers of premature infants (pMBM) obtained 3–4 weeks post-delivery was compared with MBM from mothers of term infants obtained at birth (tColostrum) and 3–4 weeks post-delivery (tMBM). The microRNA profile in lipid and skim fractions of each sample was evaluated with high-throughput sequencing. Results The expression profiles of nine microRNAs in lipid and skim pMBM differed from tMBM. Gene targets of these microRNAs were functionally related to elemental metabolism and lipid biosynthesis. The microRNA profile of tColostrum was also distinct from pMBM, but clustered closely with tMBM. Twenty-one microRNAs correlated with gestational age demonstrated limited relationships with method of delivery, but not other maternal infant factors. Conclusion Premature delivery results in a unique MBM microRNA profile with metabolic targets. This suggests preterm milk may have adaptive functions for growth in premature infants.