Objective To examine the effects of Coxsackie virus B3 (CVB3) on the NLR family, pyrin domain containing protein 3 (NLPR3) of mouse macrophages and its mechanisms. Methods RAW264.7 cells, primary mouse macrophages (bone marrow-derived macrophages or peritoneal macrophages), and short hairpin RNA (shRNA)-NLRP3 lentivirus infected RAW264.7 cells were stimulated by different dosages of CVB3. The transcript levels of NLRP3 and IL-1β were measured by quantitative real-time PCR. IL-1β in the supernatants of cell cultures was determined by ELISA. The protein level of NLRP3 was tested by Western blot analysis and the interacting proteins of NLRP3 were detected by co-immunoprecipitation (Co-IP). Results The transcript levels of NLRP3 and IL-1β were significantly up-regulated in the CVB3 stimulated RAW264.7 cells and primary mouse macrophages (bone marrow-derived macrophages or peritoneal macrophages). The expression level of NLRP3 presented CVB3-dose dependence and demonstrated the highest expression level at 6 hours