Proinflammatory cytokines such as interleukin (IL)-1β and IL-18 are known to be activated through the NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome, a multi-protein intracellular complex. MK-571 has been identified as an efflux inhibitor of multidrug resistance protein (MRP) transporters. In this study, we have revealed that MK-571 effectively suppresses the activation of the NLRP3 inflammasome in bone marrow-derived macrophages (BMDMs) by elevating intracellular cAMP levels. The accumulation of cAMP mediated by MK-571 inhibited the ATP-driven secretion of IL-1β through the ubiquitination of the NLRP3 protein. Additionally, MK-571 suppressed the nuclear factor-κB (NF-κB)-dependent priming signaling of the NLRP3 inflammasome, as evidenced by the reduction in proinflammatory cytokines and proteins as well as NF-κB-driven transcriptional activity. When administered orally, MK-571 significantly lowered mRNA levels of IL-6 and IL-1β, along with serum levels of IL-1β, in mice subjected to lipopolysaccharide/d-galactosamine treatment. These findings underline the crucial role of energy-dependent transporters MRP4 and 5 in regulating the priming and activation signals of the NLRP3 inflammasome in macrophages. Furthermore, MK-571 emerges as a promising therapeutic candidate for managing NLRP3 inflammasome-related diseases.