In this set of studies, a proteomic approach was used to investigate the protein profile of the mammary epithelial cell (MEC) through different stages of mammary development. The HC11 cell line was used to investigate protein changes between undifferentiated and differentiated MEC, which represent the pregnant and lactating states of the cells. This comparison revealed an interesting differential expression profile underscoring many recognized processes that occur in differentiated MECs, while others unveiled differences between MEC differentiation in vitro and in vivo. Primary MEC were also isolated from virgin, pregnant, and primiparous quiescent mice to compare the virgin state of the cell to the other two stages of development. These comparisons added to a previous dataset of primary isolated MEC and generated data that implied a surprising level of activity in virgin MEC relative to the other stages of development investigated. Differentially expressed proteins in the virgin and primiparous quiescent comparison also added to evidence of persisting changes occurring in the gland after a full term pregnancy that are implicated in the risk for breast cancer development. Data sets generated in the same manner from differentiating MEC were used in the development of a database to help manage the growing list of differentially expressed proteins and aid in the identification of potential interesting patterns of regulation during mammary development and differentiation.