The cell has evolved a network of diverse metal trafficking molecules, metallochaperones, that are responsible for delivery of their cargo to target molecules located in different cellular compartments. The antioxidant enzyme Cu, Zn Superoxide dismutase (Sod1) is one of these targets. It is a crucial component of the cell’s antioxidant defense system and protects it from radical oxygen assault caused as by-product of mitochondrial respiration. Sod1 has to acquire crucial post-translational modifications to become fully active: the acquisition of a structural Zn(II) ion, the binding of a catalytic Cu(I) ion, and the formation of an intramolecular disulfide bond. Copper Chaperone for Sod1 (Ccs) is responsible for proper copper delivery and Sod1 disulfide bond formation. However, the Cu(I) sources of Ccs1 for Sod1 activation have so far been poorly characterized. We demonstrate the ability of the Copper Transporter 1 (Ctr1) to donate copper directly to Ccs for Sod1 activation. Our results suggest that Sod1 activation using Ctr1 as copper source is Ccs dependent and relies on Ccs domain 1 and 3 cysteines. Both Ccs alone and Sod1-Ccs complexes can interact with Ctr1 in a copper-dependent manner, with Sod1 maturation terminating the interaction. Previous work suggested glutathione to be the major copper source for Sod1 activation; our data, however, indicates that Ccs can interact with Ctr1 directly to facilitate Sod1 activation as well. Additional experiments done suggest that Ccs is involved in all steps of Sod1 maturation. Combining spectroscopic, thermodynamic, and kinetic work, we show that our previously published Sod1-Ccs crystal structure supports a “pivot and release” model of copper transfer. Ccs is biased towards binding completely immature Sod1 and promotes Zn(II) binding. Copper gets transferred from Ccs following a thermodynamically favorable affinity gradient through an “entry site” to the Sod1 active site. Copper relay from the entry site to the active site depends solely on the formation of a critical Sod1 disulfide bond. This oxidation is dependent on the presence of oxygen and anaerobic conditions stall Sod1 maturation. The involvement of Ccs outside of its metal-chaperoning ability classifies Ccs1 as an unprecedented “dual chaperone”.