In the actinobacterium Streptomyces lividans copper homeostasis is controlled through the action of the metalloregulator CsoR. Under copper stress, cuprous ions bind to apo-CsoR resulting in the transcriptional derepression of genes encoding for copper efflux systems involving CopZ-like copper chaperones and CopA-like P-type ATPases. Whether CsoR obtains copper via a protein-protein mediated trafficking mechanism is unknown. In this study we have characterised the copper trafficking properties of two S. lividans CopZ proteins (SLI_1317 and SLI_3079) under the transcriptional control of a CsoR (SLI_4375). Our findings indicate that both CopZ-proteins have cysteine residues in the Cu(i) binding MX1CX2X3C motif with acid-base properties that are modulated for a high cuprous ion affinity and favourable Cu(i)-exchange with a target. Using electrophoretic mobility shift assays transfer of Cu(i) is shown to occur in a unidirectional manner from the CopZ to the CsoR. This transfer proceeds via a shallow thermodynamic affinity gradient and is also kinetically favoured through the modulation of the acid-base properties of the cysteine residues in the Cys2His cuprous ion binding motif of CsoR. Using RNA-seq coupled with the mechanistic insights of Cu(i) transfer between CopZ and CsoR in vitro, we propose a copper trafficking pathway for the CsoR regulon that initially involves the buffering of cytosolic copper by three CopZ chaperones followed by transfer of Cu(i) to CsoR to illicit a transcriptional response.