Abstract The NLRP3 inflammasome is a multi-protein complex that triggers the activation of inflammatory caspase-1 and the maturation of IL-1β and IL-18 in response to microbes and danger signals in the cytosol of host cells. However, how the NLRP3 inflammasome is regulated is not well defined. Here, we showed that NLRP3 undergoes tyrosine phosphorylation in macrophages upon stimulation with ATP, a NLRP3 inflammsome stimulus, and the tyrosine phosphorylation of NLRP3 correlates with its ubiquitination. Pretreating the macrophages with a specific Src kinase inhibitor inhibits NLRP3 tyrosine phosphorylation and ubiquitination, which leads to enhanced production of IL-1β. Further we found that Src family PTK Lyn phosphorylates NLRP3 at Tyr918 which facilitates its ubiquitination and proteasome-mediated degradation. Consistent with these data, NLRP3 tyrosine phosphorylation and ubiquitination is abrogated in macrophages lacking Lyn, which correlates with heightened-activation of NLRP3 inflammasome. Therefore, our data demonstrates that the Lyn-mediated tyrosine phosphorylation of NLRP3 is prerequisite for its ubiquitination, thus dampening the NLRP3 inflammasome activity.