The protein ERp57 (also known as PDIA3) is a widely distributed protein, mainly localized in the endoplasmic reticulum, where it acts as disulfide isomerase, oxidoreductase and chaperone, in concert with the lectins calreticulin (CRT) and calnexin. The ERp57/CRT complex has been detected on the cell surface and previous studies have suggested its involvement in programmed cell death. Although the ERp57-CRT complex has been characterized, little is known about its role in different cellular compartments as well as inhibitors of this interaction. We focused on the kinetic, extent and stability of the ERp57-CRT complex, using the surface plasmon resonance spectroscopy, investigating the possible role as inhibitor of the antibiotic vancomycin. Equilibrium thermodynamic data suggested that vancomycin may hinder the interaction between the two proteins and could interfere with the ERp57 conformational changes that stabilize the complex. Furthermore, by means of confocal microscopy, we evaluated the effect of the in vivo administration of vancomycin on the ERp57/CRT complex on the surface of HeLa cells. The model presented here could be used for the search of other specific inhibitors/interactors of ERp57, which can be extremely helpful to understand the biological pathways where the protein is involved and to modulate its activity.