Mutations in COCH have been associated with autosomal dominant nonsyndromic hearing loss (DFNA9) and are frequently accompanied by vestibular hypofunction. Here, we report identification of a novel missense mutation, p.F527C, located in the vWFA2 domain in members of a Korean family with late-onset and progressive hearing loss. To assess the molecular characteristics of this cochlin mutant, we constructed both wild-type and mutant cochlin constructs and transfected these into mammalian cell lines. Results of immunocytochemistry analysis demonstrated localization of the cochlin mutant in the endoplasmic reticulum/Golgi complex, whereas western blot analyses of cell lysates revealed that the mutant cochlin tends to form covalent complexes that are retained in the cell. Biochemical analyses of recombinant vWFA2 domain of cochlin carrying the p.F527C mutation revealed that the mutation increases propensity of the protein to form covalent disulfide-bonded dimers and affects the structural stability but not the collagen-affinity of the vWFA2 domain. We suggest that the instability of mutant cochlin is the major driving force for cochlin aggregation in the inner ear in DFNA9 patients carrying the COCH p.F527C mutation.