Manganese (Mn) is essential for many enzymatic processes in the brain; however, Mn overload can lead to neurotoxicity and behavioral deficits. The metal transporter ZIP14 (SLC39A14) plays a key role in systemic Mn homeostasis, yet its role in brain Mn homeostasis remains unclear. This dissertation investigates ZIP14’s roles in brain endothelial cells and astrocytes.The blood-brain barrier (BBB), comprised of polarized endothelial cells, tightly regulates metals in and out of the brain. However, ZIP14’s precise localization and role in BBB endothelial cells have yet to be defined. Here, we show in vivo ZIP14 expression in BBB endothelial cells, which upregulates following Mn supplementation. Expansion microscopy revealed a shift in ZIP14 localization from equal apical-basolateral distribution to predominantly basolateral after Mn exposure. Endothelial-specific Zip14 KO (EKO) mice exhibited impaired Mn efflux from the brain and increased brain Mn accumulation after Mn supplementation. In vitro studies using EKO-derived primary endothelial cells and ZIP14-overexpressing hCMEC/D3 cells confirmed that ZIP14 facilitates basolateral-to-apical Mn transport. These results demonstrate that ZIP14 is critical for brain Mn clearance, We also observed ZIP14 expression in primary astrocytes isolated from wild-type mice. Using astrocytes derived from astrocyte-specific Zip14 knockout mice, we demonstrated that ZIP14 mediates Mn uptake into astrocytes. Additionally, ZIP14 was partially localized to the mitochondria, and its deletion in astrocytes protected against the Mn-induced mitochondrial dysfunction, as evidenced by the preserved mitochondrial morphology, improved mitochondrial respiration and metabolic activity compared to controls. Collectively, these studies establish ZIP14 as a key mediator of brain Mn homeostasis, with cell-specific roles: promoting Mn clearance from the BBB and contributing to Mn toxicity in astrocytes. Our findings advance the understanding of ZIP14’s functions in Mn neurotoxicity mechanisms and may serve as a potential therapeutic target to mitigate Mn-induced neurotoxicity.