NLRP3 is an intracellular pattern-recognition receptor that senses many different activators including bacterial toxins and endogenous danger signals. Upon activation, the NLRP3 inflammasome is formed, leading to the secretion of pro-inflammatory cytokines and cell death. Because of its involvement in many diseases such as atherosclerosis and Alzheimer’s Disease, a better understanding of NLRP3 regulation may help to shape therapeutic approaches. The aim of this study was to identify post-translational modifications on NLRP3 and their impact on inflammasome activation. We applied mass spectrometry of NLRP3 to identify post-translational modifications. We identified ubiquitinated and phosphorylated residues. To analyze the function of the modifications, we mutated the residues in NLRP3 expression plasmids and used them for reconstitution of NLRP3-deficient macrophages or overexpression studies in HEK293T cells. Ubiquitinylated residues were found in the NACHT and LRR domains of NLRP3 and mutation of these residues resulted in hyperactive NLRP3. In addition, mutation of one of the phosphorylated residues to a phosphomimetic residue showed reduced IL-1beta release and caspase-1 cleavage upon NLRP3 inflammasome activation, whereas mutation to alanine had no effect. Further studies in HEK293T cells found that the recruitment of the downstream adaptor ASC as well as the self-interaction of NLRP3 were reduced with the phosphomimetic residue. Thus, NLRP3 is likely regulated by post-translational modification and needs to be deubiquitinylated and possibly dephosphorylated before activation. Knowledge about the modifications and the pathways involved may help shape novel strategies for treating diseases that involve NLRP3.