Protein folding in the endoplasmic reticulum is often associated with the formation of native disulfide bonds. Their primary function is to stabilize the folded structure of the protein, although disulfide bond formation can also play a regulatory role. Native disulfide bond formation is not trivial, so it is often the rate-limiting step of protein folding both in vivo and in vitro. Complex coordinated systems of molecular chaperones and protein folding catalysts have evolved to help proteins attain their correct folded conformation. This includes a family of enzymes involved in catalyzing thiol-disulfide exchange in the endoplasmic reticulum, the protein disulfide isomerase (PDI) family. There are now 17 reported PDI family members in the endoplasmic reticulum of human cells, but the functional differentiation of these is far from complete. Despite PDI being the first catalyst of protein folding reported, there is much that is still not known about its mechanisms of action. This review will focus on the interactions of the human PDI family members with substrates, including recent research on identifying and characterizing their substrate-binding sites and on determining their natural substrates in vivo.