The inflammasome is a critical molecular complex that activates IL-1 driven inflammation in response to pathogen- and danger-associated signals. Germline mutations in the inflammasome sensor, NLRP1 cause Mendelian systemic autoimmunity and skin cancer susceptibility, but its endogenous regulation remains less understood. Here we use a proteomics screen to uncover dipeptidyl dipeptidase, DPP9 as a novel interacting partner with human NLRP1 and a related inflammasome regulator, CARD8. DPP9 functions as an endogenous inhibitor of NLRP1 inflammasome in diverse primary cell types from human and mice. DPP8/9 inhibition via small molecule drugs and CRISPR/Cas9-mediated genetic deletion specifically activate human NLRP1 inflammasome, leading to ASC speck formation, pyroptotic cell death and secretion of cleaved IL-1beta. Mechanistically, DPP9 interacts with a unique auto-proteolytic domain (FIIND) found in NLRP1 and CARD8. This scaffolding function of DPP9 and its catalytic activity act synergistically to maintain NLRP1 in its inactive state and repress downstream inflammasome activation. We further identified a single patient-derived germline missense mutation in NLRP1 FIIND domain that abrogates DPP9 binding, leading to inflammasome hyper-activation seen in the Mendelian auto-inflammatory disease AIADK. These results unite recent findings on the regulation of murine Nlrp1b by Dpp8/9 and uncover a new regulatory mechanism for the NLRP1 inflammasome in primary human cells. Our results further suggest that DPP9 could be a multi-functional inflammasome regulator involved in human auto-inflammatory diseases.