NLRP3 is activated by many stimuli and its dysfunction is involved in various autoinflammatory diseases. NLRP3 aggregates preceding the release of its autoinhibitory conformation where scaffold proteins mediating aggregation are proposed. How NLRP3 releases auto-repression remains elusive. Here, we report that activating molecule in Beclin1-regulated autophagy protein 1 (AMBRA1) binds NLRP3 to scaffold and allosterically activate NLRP3. AMBRA1 engages NLRP3’s transition LRR and HD2 subdomains via its β-propeller to destabilize the closed inactive NLRP3 conformation, facilitating ATP binding and transition to the active state, which is similar to Apaf1-scaffolded apoptosome assembly. AMBRA1 deficiency in cells and in mice impairs NLRP3 activation and inflammatory responses in mouse models. Blocking AMBRA1-NLRP3 interaction by nanobodies inhibits NLRP3 activation, underscoring the therapeutic potential of targeting this interaction. This study implies a conserved activating mechanism shared by both apoptotic and pyroptotic cell death and β-propeller-containing partners like AMBRA1 are required for the activation of NLRPs.