ER protein 57 (ERp57), a thiol isomerase secreted from vascular cells, is essential for complete thrombus formation in vivo, but other extracellular ERp57 functions remain unexplored. Here, we employed a kinetic substrate-trapping approach to identify extracellular protein substrates of ERp57 in platelet-rich plasma. MS-based identification with immunochemical confirmation combined with gene ontology enrichment analysis revealed that ERp57 targets, among other substrates, components of the lectin pathway of complement activation: mannose-binding lectin, ficolin-2, ficolin-3, collectin-10, collectin-11, mannose-binding lectin-associated serine protease-1, and man-nose-binding lectin-associated serine protease-2. Ficolin-3, the most abundant lectin pathway initiator in humans, circulates as disulfide-linked multimers of a monomer. ERp57 attenuated ficolin-3 ligand recognition and complement activation by cleaving intermolecular disulfide bonds in large ficolin-3 multimers, thereby reducing multimer size and ligand-binding affinity. We used MS to identify the disulfide-bonding pattern in ficolin-3 multimers and the disulfide bonds targeted by ERp57 and found that Cys6 and Cys23 in the N-terminal region of ficolin-3 form the intermolecular disulfide bonds in ficolin-3 multimers that are reduced by ERp57. Our results not only demonstrate that ERp57 can negatively regulate complement activation, but also identify a control mechanism for lectin pathway initiation in the vasculature. We conclude that extensive multimerization in large ficolin-3 multimers leads to a high affinity for ligands and strong complement-activating potential and that ERp57 suppresses complement activation by cleaving disulfide bonds in ficolin-3 and reducing its multimer size.