Abstract: Mitochondria contain two biological membranes. Although reducing agents can diffuse from the cytosol into the intermembrane space (IMS) between the outer and inner mitochondrial membranes, the IMS has a dedicated disulfide relay system to introduce disulfide bonds into mainly small and soluble proteins. This system consists of two essential proteins, a disulfide carrier Tim40/Mia40 and a flavin-dependent sulfhydryl oxidase Erv1, high-resolution structures that have recently become available. Tim40/Mia40 transfers disulfide bonds to newly imported IMS proteins by dithiol/disulfide exchange reactions involving mixed disulfide intermediates. Tight folding by introduction of disulfide bonds prevents egress of these small IMS proteins, resulting in their selective retention in the compartment. After disulfide transfer from Tim40/Mia40 to substrate proteins, Tim40/Mia40 is reoxidized again by Erv1, which is then oxidized by electron transfer to either cytochrome c or molecular oxygen. Here we review the recent advancement of the knowledge on the mechanism of the disulfide relay system in the mitochondrial IMS, especially shedding light on the structural aspects of its components.