Since protein disulfide isomerase (PDI) was first described in 1963, researchers have shown conclusively that PDI and sibling proteins are quintessential for thrombus formation. PDI, endoplasmic reticulum protein (ERp)5, ERp57, and ERp72 are released from platelets and vascular cells and interact with integrin αIIbβ3 on the outer surface of platelets. At the cell surface they influence protein folding and function, propagating thrombosis and maintaining hemostasis. TMX1, which is a transmembrane thiol isomerase, is the first family member shown to negatively regulate platelets. Targets of thiol isomerases have been identified, including integrin α2β1, Von Willebrand Factor, GpIbα, nicotinamide adenine dinucleotide phosphate oxidase (Nox)-1, Nox-2, and tissue factor, all of which are pro-thrombotic, and several of which are on the cell surface. In spite of this, PDI can paradoxically catalyze the delivery of nitric oxide to platelets, which decrease thrombus formation. Although the overall effect of PDI is to positively regulate platelet activation, it is still unclear how thiol isomerases function in pro-thrombotic states, such as obesity, diabetes, and cancer. In parallel, there has been a surge in the development of novel thiol isomerase inhibitors, which display selectivity, potency and modulate thrombosis and hemostasis. The availability of selective thiol isomerase inhibitors has culminated in clinical trials, with promising outcomes for the prevention of cancer-associated thrombosis. Altogether, thiol isomerases are perceived as an orchestrating force that regulates thrombus development. In the current review, we will explore the history of PDI in cardiovascular biology, detail known mechanisms of action, and summarize known thiol isomerase inhibitors.