Human butyrophilin (BTN) expression in milk fat globule (MFGM) was evaluated using two dimensional electrophoresis (2-DE) as the separation technique, and peptide mass mapping by matrix-assisted laser desorption/ionisation-mass spectrometry (MALDI-MS) as the identification tool. Since milk composition changes throughout lactation time, 2-DE maps in the pH range 4-7 of colostral MFGM and mature MFGM were compared, showing only slight differences in BTN spot distribution. The BTN gene family codes for seven proteins (BTN, BTN2A1, BTN2A2, BTN2A3, BTN3A1, BTN3A2, BTN3A3), their presence in human tissues has to date been evaluated only at a transcriptional level. Among 70 spots, analyzed and identified by MALDI-MS, 13 spots were identified as BTN spots and only one as a fragment of BTN2A1. BTN was present in multiple glycoforms, and two smaller BTN forms of about 45 kDa were also identified. We propose an array of BTNs on human MFGM, which could provide breast-fed infants with immune molecules during the neonatal period.