Organophosphate esters (OPEs) are used in consumer products as flame retardants and plasticizers. Little is known, however, about the occurrence and profiles of OPEs in human milk. In this study, 14 OPEs were measured in 100 breast milk samples collected from the United States during the period of 2009–2012, using high-performance liquid chromatography and tandem mass spectrometry. The sum concentrations of 14 OPEs in human milk ranged from 0.670 to 7.83 ng/mL, with a mean value of 3.61 ng/mL. The highest mean concentration was found for tris-2-butoxyethyl phosphate (TBOEP, 1.44 ± 0.789 ng/mL), followed by tri-iso-butyl phosphate (TIBP, 0.569 ± 0.272 ng/mL) and tri-n-butyl phosphate (TNBP, 0.539 ± 0.265 ng/mL), which were the dominant OPEs found in breast milk at detection frequencies of >80%. No significant differences were observed between various maternal/infant characteristics and OPE concentrations (p > 0.05), except for TBOEP, for which the median concentrations in Hispanic mothers (0.765 ng/mL) were 2 times lower than those in non-Hispanic mothers (1.48 ng/mL) (p < 0.05). On the basis of the recommended daily milk ingestion rate, the average and the highest daily intakes of total OPEs were calculated to be in the range of 300–542 and 504–911 ng (kg of body weight)⁻¹ day^–1, respectively. The estimated daily intakes of OPEs did not exceed the current reference doses. Our study establishes baseline data for OPE exposure in breast-fed American children.