Lactation, the physiological condition during which milk is produced for the purpose of providing nutrition to the offspring, features numerous anatomical, cellular, physiological, and behavioral adaptations, which affect the central nervous system as well as the mammary gland. Traditionally, lactation comprise of the sequential phases of lactogenesis I, the onset of milk synthesis without secretion during late pregnancy; lactogenesis II, the onset of active milk secretion shortly after parturition; and lactogenesis III (galactopoiesis), the maintenance of lactation [1–3]. Milk ejection, or letdown, refers to the process by which milk is removed from the mammary gland during lactation by the nursing offspring [1,2,4]. All aspects of lactation are influenced by multiple, interacting endocrine and paracrine messengers, including ovarian and adrenal steroids and insulin [1,2]. However, prolactin (PRL), released from the anterior pituitary gland and critical for milk synthesis and secretion, and oxytocin (OT), released from the posterior pituitary and required for milk ejection, are arguably the most important hormonal regulators of lactation, and their patterns of secretion, physiological actions and neuroendocrine regulation will be the focus of this review. Table 6.1 shows some actions of these and other hormones, which are exerted within the mammary gland and important for lactation.