The NLRP3 inflammasome, which regulated a proinflammatory programmed cell death form termed pyroptosis, is involved in the pathological process of various human diseases, such as multiple sclerosis, type 2 diabetes, and gout. Thus, compounds inhibiting activation of the NLRP3 inflammasome can be promising treatments for these diseases. In this study, we conducted a phenotypic screening against NLRP3-dependent pyroptosis and discovered the hit compound 1, which showed moderate antipyroptotic activity. Chemistry efforts to improve potency of 1 resulted in a novel compound 59 (J114), which exhibited a half-maximal inhibitory concentration (IC₅₀) of 0.077 ± 0.008 μM against cell pyroptosis. Interestingly, unlike all pyroptosis inhibitors currently reported, the activity of J114 showed significant differences in human- and mouse-derived cells. The IC₅₀ of J114-mediated inhibition of IL-1β secretion by human THP-1 macrophages was 0.098 μM, which was nearly 150-fold and 500-fold more potent than that of J774A.1 (14.62 μM) and bone marrow-derived macrophages (BMDMs) (48.98 μM), respectively. Further studies showed that J114 displayed remarkable inhibitory activity against NLRP3- and AIM2-but not NLRC4-dependent activation of caspase-1 and the release of IL-1β in human THP-1 macrophages. Mechanistically, J114 disturbed the interaction of NLRP3 or AIM2 with the adaptor protein ASC and inhibited ASC oligomerization. Overall, our study identified a unique molecule that inhibits NLRP3 and AIM2 inflammasome activation and has species differences, which is worthy of further research to understand the differential regulation of the NLRP3 and AIM2 inflammasomes in humans and mice.