The lactating mammary gland is comprised of multiple cells types which tightly coordinate the accumulation, production and secretion of milk components including essential metals such as zinc (Zn). Our previous studies in animal and cell models implicated the Zn transporter Zip3 (Slc39a3) in mammary gland Zn acquisition. Herein we investigated this hypothesis directly by utilizing Zip3-null mice. Our data verify that Zip3 is expressed in secretory mammary cells; however, Zip3 does not play a major role in Zn import from the maternal circulation. Importantly, the primary localization of Zip3 was primarily associated with the luminal membrane of the secretory mammary cells. Consistent with this localization, Zn transfer studies using ⁶⁵Zn revealed that Zn retention in the secreted milk pool and milk Zn concentration was higher in Zip3-null compared with wild-type mice. Although total mammary gland Zn concentration was not altered, Zip3-null mice also had altered mammary tissue architecture, increased number of apoptotic cells and reduced mammary gland weight implicating subtle changes in Zip3-mediated intracellular Zn pools in apoptosis regulation. Taken together our data indicate that Zip3 does not participate in the acquisition of Zn from maternal circulation for secretion into milk but in contrast primarily plays a role in the reuptake and cellular retention of Zn in the mammary gland from the previously secreted milk pool thus regulating cellular function.