ABSTRACT Pyrin is a pattern-recognition receptor in phagocytes that triggers caspase-1 inflammasome assembly in response to bacterial toxins and effectors that inactivate RhoA. Pyrin contains oligomerization domains and is negatively regulated by phosphorylation of two residues, S205 and S241 (murine) or S208 and S242 (human), via the kinases PKN1/2, which are activated by RhoA. Familial Mediterranean Fever (FMF) is caused by the phagocyte production of pyrin gain of function variants, which have a lower threshold for inflammasome assembly upon RhoA-PKN axis inhibition. Inactivation of the RhoA-PKN axis removes negative regulation but a phosphoprotein phosphatase (PPP) is needed to positively regulate pyrin. No PPP that dephosphorylates pyrin has been identified, oligomerization of murine pyrin has not been studied, and the phosphorylation status of oligomeric pyrin is unknown. We used murine macrophages and FMF patient’s monocytes combined with the use of bacterial agonists and chemical inhibitors, native PAGE, phospho-specific antibodies, and siRNA knockdowns to determine if a PPP positively regulates oligomeric pyrin. Results with broadly specific inhibitors indicate that PPP activity is required to dephosphorylate murine and human pyrin in wild-type or FMF patient’s phagocytes. Findings from native PAGE show that murine pyrin is oligomeric and phosphorylated on S205 prior to RhoA inactivation. PPP inhibitors cause reduced mobility of murine pyrin on native PAGE and increased phosphorylation of S242 in human pyrin, suggesting a PPP constitutively counterbalances PKN to regulate second-site phosphorylation. Data from siRNA knockdown experiments implicate PP2A in dephosphorylation of S205 and positive regulation of pyrin in response to RhoA inactivation. IMPORTANCE Pyrin, a unique cytosolic receptor, initiates inflammatory responses against RhoA-inactivating bacterial toxins and effectors like Yersinia’s YopE and YopT. Understanding pyrin regulation is crucial due to its association with dysregulated inflammatory responses, including Familial Mediterranean Fever (FMF), linked to pyrin gene mutations. FMF mutations historically acted as a defense mechanism against plague. Negative regulation of pyrin through PKN phosphorylation is well established, with Yersinia using the YopM effector to promote pyrin phosphorylation and counteract its activity. This study highlights the importance of phosphoprotein phosphatase activity in positively regulating pyrin inflammasome assembly in phagocytic cells of humans and mice. Oligomeric murine pyrin has S205 phosphorylated before inflammasome assembly, and this study implicates the dephosphorylation of murine pyrin S205 by two catalytic subunits of PP2A in macrophages. These findings offer insights for investigating the regulation of oligomeric pyrin and the balance of kinase and phosphatase activity in pyrin-associated infectious and autoinflammatory diseases.