The NLRP3 inflammasome interacts with the adaptor ASC to activate caspase-1 for the maturation of pro-IL-1β into active IL-1β. The formation and activity of NLRP3 inflammasome is regulated through mechanisms not fully elucidated. It was recently demonstrated that leucine-rich repeat Flightless-I-interaction protein 2 (LRRFIP2) and Flightless-I (FliI) negatively regulate NLRP3 inflammasome activity. It was also showed that FliI is a pseudosubstrate and inhibitor of caspase-1. Since FliI is an actin-remodeling protein, we addressed the question of a role of actin in the regulation NLRP3 inflammasome in human macrophages (THP-1). The depolymerization of filamentous actin (F-actin) in globular actin (G-actin) significantly increased the production of mature IL-1β while the stabilization of F-actin decreased IL-1β production. These results suggest that activation of NLRP3 inflammasome depends on the actin polymerization state but not on the active polymerization process. The silencing of LRRFIP2 and FliI by lentiviral-based shRNA transduction showed that LRRFIP2 and FliI were required for co-localization of NLRP3 on F-actin in response to ATP or Nigericin. Thus, FliI-mediated caspase-1 inhibition involves the localization of NLRP3 inflammasome on F-actin leading to the diminution of caspase-1 activation and in turn to decreased IL-1β production. The present data demonstrate that in addition to directly inhibiting caspase-1, FliI together with LRRFIP2 allow the location of the NLRP3 inflammasome on F-actin, further inhibiting caspase-1 activation and IL-1β production. Our results unveil a new function of actin and a dual function of FliI in the regulation of NLRP3 inflammasome activity strengthening the importance of cytoskeleton in the regulation of inflammation.