AbstractStreptococcus suis serotype 2 (SS2) is an important zoonotic pathogen that induces an NLRP3-dependent cytokine storm. NLRP3 inflammasome activation triggers not only an inflammatory response but also pyroptosis. However, the exact mechanism underlying S. suis-induced macrophage pyroptosis is not clear. Our results showed that SS2 induced the expression of pyroptosis-associated factors, including lactate dehydrogenase (LDH) release, propidium iodide (PI) uptake and GSDMD-N expression, as well as NLRP3 inflammasome activation and IL-1β secretion. However, GSDMD deficiency and NLRP3 inhibition using MCC950 attenuated the SS2-induced expression of pyroptosis-associated factors, suggesting that SS2 induces NLRP3-GSDMD-dependent pyroptosis. Furthermore, RACK1 knockdown also reduced the expression of pyroptosis-associated factors. In addition, RACK1 knockdown downregulated the expression of NLRP3 and Pro-IL-1β as well as the phosphorylation of P65. Surprisingly, the interaction between RACK1 and P65 was detected by co-immunoprecipitation, indicating that RACK1 induces macrophage pyroptosis by mediating the phosphorylation of P65 to promote the transcription of NLRP3 and pro-IL-1β. Similarly, NEK7 knockdown decreased the expression of pyroptosis-associated factors and ASC oligomerization. Moreover, the results of co-immunoprecipitation revealed the interaction of NEK7-RACK1-NLRP3 during SS2 infection, demonstrating that NEK7 mediates SS2-induced pyroptosis via the regulation of NLRP3 inflammasome assembly and activation. These results demonstrate the important role of RACK1 and NEK7 in SS2-induced pyroptosis. Our study provides new insight into SS2-induced cell death.