Heat stress (HS) reduces cows’ milk production, resulting in a significant economic loss for the dairy industry. During lactation, HS lowers milk yield by 25–40% with half of the decrease in milk synthesis due to factors unrelated to feed intake. In vitro studies indicate that primary bovine mammary epithelial cells display greater rates of programmed cell death when exposed to high ambient temperature, which may lead to a decrease in total number of milk synthetic cells in the mammary gland (MG) and partially explain the lower milk production of lactating cows under HS. The function of mammary cells is also altered by HS. In response to HS, mammary cells display higher gene expression of heat shock proteins, indicating a need for cytoprotection from protein aggregation and degradation. Further, HS results in increased gene expression but similar protein expression of mammary epithelial junction proteins, and doesn’t substantially influence the integrity of mammary epithelium, indicating an effort to maintain cell-to-cell junction by synthesizing more proteins to compensate for protein loss by HS. Bovine mammary epithelial cells also have reduced gene expression of proteins involved in milk synthesis suggesting that HS directly reduces milk synthetic capacity of MG. During the dry period, HS negatively affects MG development by reducing mammary cell proliferation before parturition, resulting in a dramatic decrease in milk production in the subsequent lactation. In addition to mammary growth, MG of the HS cow has reduced protein expression of autophagy proteins in the early dry period, suggesting HS influences mammary involution. Emerging evidence also indicates that heifers born to late gestation HS cows have lower milk production during their first lactation, implying that the maternal environment may alter MG development of the offspring. It is not clear if this is due to a directly epigenetic modification of prenatal MG development by maternal HS. More research is needed to elucidate the impact of HS on MG development and function.