Marginal intake of zinc and vitamin A is common during lactation and a deficiency of one micronutrient can result in a secondary deficiency of the other. However, the resistance of milk zinc (Zn) concentration to changes in dietary Zn or vitamin A indicates tight regulation of mammary gland Zn transport. Although several mammalian proteins have been identified and implicated in Zn transport, the mechanisms responsible for mammary gland Zn transport and their regulation by dietary Zn and vitamin A are unknown. In this study, we identified mammary gland Zn transporters and determined effects of marginal Zn and vitamin A intakes on their levels. Rats were fed a control [25 mg Zn/kg, 4 retinol equivalents (RE)/g], a low Zn (10 mg Zn/kg), a low vitamin A (0.4 RE/g), or a low Zn (10 mg Zn/kg) and vitamin A (0.4 RE/g) diet throughout lactation. ZnT-1, ZnT-2 and ZnT-4 were identified in the mammary gland and localized to the serosal membrane (ZnT-1) or intracellularly (ZnT-2 and ZnT-4) by immunostaining. Rats fed a low Zn or low vitamin A diet had lower ZnT-1 protein and higher ZnT-4 mRNA expression and protein levels compared with controls. There was a significant interaction between dietary Zn and vitamin A on zinc transporter mRNA expression and protein levels. Although total mammary gland Zn was not affected, mammary gland metallothionein levels were lower in rats fed low Zn and higher in rats fed low vitamin A, suggesting different mechanisms regulating zinc transporter levels. These results indicate that milk Zn level is maintained through coordinated regulation of mammary gland zinc transporters and documents an effect of vitamin A on zinc homeostasis at the molecular level during lactation.