Metastasis and recurrence of breast cancer remain major causes of patient mortality and there is an ongoing need to identify new therapeutic targets relevant to tumour invasion. Protein disulphide isomerase A3 (PDIA3) is a disulphide oxidoreductase and isomerase of the endoplasmic reticulum that has known extracellular substrates and has been correlated with aggressive breast cancers. We show that either prior PDIA3-inhibition by the disulphide isomerase inhibitor 16F16 or depletion of heparin-binding proteins strongly reduces the activity of conditioned medium (CM) of MDA-MB-231 human breast cancer cells to support pro-migratory cell spreading and F-actin organisation by newly adherent MDA-MB-231 cells. Quantitative proteomics to investigate effects of 16F16 inhibition on heparin-binding proteins in the CM of MDA-MB-231 cells identified 80 proteins reproducibly decreased >2-fold (at q <0.05) after 16F16 treatment. By Gene Ontology analysis, many of these have roles in extracellular matrix structure and function and cell adhesion; ribosomal proteins that also correlate with extracellular vesicles were also identified. Protein-protein interaction analysis showed that many of the extracellular proteins have known network interactions with each other. The predominant types of disulphide-bonded domains in the extracellular proteins contained beta-hairpin folds, with the knottin fold the most common. From human breast cancer datasets, the extracellular proteins were found to correlate specifically with the basal subtype of breast cancer and their high expression in tumours correlated with reduced distant metastasis-free survival. These data provide new evidence that PDIA3 may be a relevant therapeutic target to alter properties of the ECM-associated microenvironment in basal breast cancer.