It is discussed that specific amino acids (AAs) have functional roles in early life. Understanding the AA composition in human milk (HM) during lactation assists in specifying these roles. To this end we assessed the levels of free AAs (FAAs), total AAs (free and bound, TAAs) and protein levels in HM in the first 6 months of lactation, and evaluated possible associations with infant gender. HM samples of 25 healthy Dutch mothers participating in the PreventCD study were collected monthly during the first 6 months of lactation. Of the participating mothers, 12 gave birth to a boy and 13 gave birth to a girl. Analyses of the HM samples revealed that levels of free glutamate, glutamine, aspartate, glycine, and serine significantly increased during months 1–3 of lactation, both in absolute sense and relative to TAA levels. Evaluation of gender differences by mixed model analyses revealed an association between female infant gender and higher protein content (p = 0.0465) and TAA content (p = 0.0362) in HM during the first 3 months of lactation. Furthermore, there was a tendency for an association of male infant gender with higher levels of free glutamine (p = 0.0948) in HM during the first 3 months of lactation. These results show that FAA, TAA and protein levels in HM display a time-specific occurrence during lactation. Moreover, although confirmation is necessary in view of the small sample size, this study indicates that the AA composition in HM shows differential effects of the infant’s sex.