Yeast two hybrid experiments identified alpha2-Heremans-Schmid glycoprotein (AHSG, human fetuin A) as a binding partner for calpain domain III (DIII). The tandem DIIIs of calpain-10 interacted under the most selective culture conditions, but DIIIs of m-calpain, calpain-3, and calpain-5 also interacted under less stringent selection. DIIIs of µ-calpain, calpain-6, and the tandem DIII-like domains of the Dictyostelium Cpl protein did not interact with AHSG in the yeast two-hybrid system. Bovine fetuin A stabilized proteolytic activity of purified m-calpain incubated in the presence of mM calcium chloride, and prevented calcium-dependent m-calpain aggregation. Consistent with the yeast two-hybrid studies, fetuin A neither stabilized µ-calpain nor prevented its aggregation. Confocal immunofluorescence microscopy of scratch-damaged L6 myotubes demonstrated accumulation of m-calpain at the wound site in association with the membrane repair protein, dysferlin. m-Calpain also co-localized with fluorescein-labeled fetuin A at the wound site. The effect of fetuin A on calpain-mediated plasma membrane resealing was investigated using fibroblasts from Capns1-/- and Capns1+/+ mouse embryos. Capns1 encodes the small non-catalytic subunit that is required for the proteolytic function of m- and µ-calpains. Thus, Capns1-/- fibroblasts do not express these calpains in active form. Fetuin A increased resealing of scrape-damaged wild-type, but not Capns1-/- fibroblasts. These studies identify fetuin A as a potential extracellular regulator of m-calpain at nascent sites of plasma membrane wounding.