Prolactin (PRL) is a single chain polypeptide of 199 amino acids with a molecular weight of about 24,000 daltons. Its structure is similar to that of growth hormone and placental lactogen. The molecule is folded due to the activity of three disulfide bonds. Significant heterogeneity of the molecule has been described, thus bioassays and immunoassays can give different results due to differing glycosylation, phosphorylation, sulfation, as well as degradation. The non-glycosylated form of prolactin is the dominant form of prolactin that is secreted by the pituitary gland. Prolactin is a peptide hormone primarily associated with lactation. In breastfeeding, the infant suckling the teat stimulates the production of prolactin, which fills the breast with milk (lactogenesis) in preparation for the next feed. Oxytocin, a similar hormone, is also released, which triggers milk let-down.

Prolactin has many effects: The most important of which is to stimulate the mammary glands to produce milk (lactation). Increased serum concentrations of prolactin during pregnancy cause enlargement of the mammary glands of the breasts and increases the production of milk. However, the high levels of progesterone during pregnancy act directly on the breasts to stop ejection of milk. It is only when the levels of this hormone fall after childbirth that milk ejection is possible. Sometimes, newborn babies (males as well as females) secrete a milky substance from their nipples. This substance is commonly known as Witch's milk. This is caused by the fetus being affected by prolactin circulating in the mother just before birth, and usually stops soon after birth.  Another effect, recently discovered by the University of Paisley and the ETH Zürich, is to provide the body with sexual gratification after sexual acts. The hormone represses the effect of dopamine, which is responsible for sexual arousal, thus causing the male's refractory period. The amount of prolactin can be an indicator for the amount of sexual satisfaction and relaxation. Unusually high amounts are suspected to be responsible for impotence and loss of libido.  Prolactin has been found to stimulate proliferation of oligodendrocyte precursor cells. These cells differentiate into oligodendrocytes, the cells responsible for the formation of myelin coatings on axons in the central nervous system.

Other possible functions of prolactin include the surfactant synthesis of the fetal lungs at the end of the pregnancy and immune tolerance of the fetus by the maternal organism during pregnancy.