Milk fat globule membrane (MFGM) contains proteins derived from the apical membrane of secreting epithelial cells of the mammary gland. Between 2–4% of total human milk protein content is associated with the fat globule fraction, as MFGM proteins. While MFGM proteins have very low classical nutritional value, they play important roles in various cell processes and defence mechanisms for the newborn. To date, fewer than 30 human MFGM proteins have been identified and characterized, either by immunological methods or by Edman sequencing and mass spectrometry. This study aimed to update the structural proteome of human colostral MFGM proteins and to create an annotated two‐dimensional electrophoresis (2‐DE) MFGM protein database available on‐line. More than one hundred 2‐DE spots derived from human colostral MFGM proteins were investigated by matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry and proteins were identified by three different software packages available on the web (PeptIdent, MS‐Fit and ProFound); uncertain identifications were solved by nanoelectrospray ionization‐ion trap mass spectrometry using SEQUEST software.