Studies have previously shown that mutant calreticulin (CALR), found in a subset of patients with myeloproliferative neoplasms (MPNs), interacts with and subsequently promotes the activation of the thrombopoietin receptor (MPL). However, the molecular mechanism behind the activity of mutant CALR remains unknown. Here we show that mutant, but not wild-type, CALR interacts to form a homomultimeric complex. This intermolecular interaction among mutant CALR proteins depends on their carboxyl-terminal domain, which is generated by a unique frameshift mutation found in patients with MPN. With a competition assay, we demonstrated that the formation of mutant CALR homomultimers is required for the binding and activation of MPL. Since association with MPL is required for the oncogenicity of mutant CALR, we propose a model in which the constitutive activation of the MPL downstream pathway by mutant CALR multimers induces the development of MPN. This study provides a potential novel therapeutic strategy against mutant CALR-dependent tumorigenesis via targeting the intermolecular interaction among mutant CALR proteins.