During lactation, functionally differentiated mammary epithelial cells convert circulating nutrients into various milk components, providing all essential nutrients for the growth and development of mammal neonates. One of the major milk components is milk protein, which includes the casein and whey proteins. Regulation of milk protein gene expression is dependent on hormonal and developmental cues that modulate the activity of specific transcription factors and change the chromatin structure in mammary epithelial cells. Understanding the underlying mechanisms involved in mammary-specific milk protein gene regulation will help improve the yield, quality, and efficiency of milk production and identify important signaling factors and pathways involved in mammary development, differentiation, lactation, and disease. In this review we first review advances in the understanding of the regulatory mechanisms of milk protein genes by hormones, growth factors, and the extracellular matrix, with a focus on transcriptional regulation. We then discuss the relationship between chromatin structure and milk protein gene expression from an epigenetic perspective. Finally, we summarize recent achievements using the mammary gland as a bioreactor for producing pharmaceutical proteins for human use.