Abstract Background. Pyrin/TRIM20 is expressed in the neutrophils and monocytes/macrophages and regulates caspase-1 activation and interleukin-1β maturation. Although the mutations in the PRY/SPRY domain of pyrin cause familial Mediterranean fever (FMF), the mechanism of how mutated pyrin provokes excessive inflammation in FMF patients is not well understood. This study was undertaken to explore the new pyrin PRY/SPRY domain-binding protein and to investigate how the interaction affected the pyrin function.Methods. We carried out the yeast two-hybrid screening for pyrin PRY/SPRY domain-binding proteins. Then, protein interactions were investigated using Lenti-X 293T cells expressing pyrin-associated proteins by co-immunoprecipitation analysis. We also used human embryonic kidney (HEK) 293 cells expressing pyrin-associated proteins and human neutrophils stimulated with monosodium urate crystal for immunofluorescence staining analysis.Results. β2-Microglobulin (β2MG) was identified as the novel pyrin ligand binding to the PRY/SPRY domain. β2MG was co-localized with pyrin not only in the HEK293 cells overexpressing these proteins but also in the stimulated human neutrophils in the speck-like structures. The pyrin-β2MG interaction triggered the binding of pyrin and proline-serine-threonine phosphatase interacting protein 1 (PSTPIP1) and then the subsequent recruitment of apoptosis-associated speck-like protein containing caspase recruitment domain (ASC). Caspase-1 p20 subunit, produced by pyrin inflammasome, also interacted with the pyrin PRY/SPRY domain and inhibited the pyrin-β2MG interaction. FMF-associated pyrin mutation M694V did not affect pyrin-β2MG interaction but weakened this inhibition.Conclusions. Our findings suggest that β2MG functions as the pyrin ligand inducing pyrin inflammasome formation and that the FMF-associated pyrin mutations weakened negative feedback of caspase-1 p20 subunit.