NLRP3 inflammasome is one of the most well-known inflammasomes in mammals, which plays critical roles in innate immunity. However, knowledge about this inflammasome in non-mammalian species, especially in teleost fish, remains rarely known. Herein, we established an Edwardsiella piscicida-head-kidney macrophages (HKMs) infection model in Japanese flounder, and found a robust caspase-1 activation and IL-1β maturation. To characterize the upstream receptor, we established a bioinformatic screening analysis, and found an NLRP3 homolog (JfNLRP3) from Japanese flounder, which shares an overall conservative structure architecture to human NLRP3. Moreover, the JfNLRP3 can assemble JfASC through PYD-PYD domain interaction and trigger JfCaspase-1 activation and JfIL-1β maturation. Meanwhile, the classical inflammasome activation stimulators, including nigericin, ATP or MSU, can trigger the JfCaspase-1 activation and JfIL-1β maturation in Japanese flounder HKMs. During intraperitoneal infection of E. piscicida in Japanese flounder, we found a dynamic up-regulated transcription of JfNLRP3 and JfCaspase-1 in vivo. Furthermore, knockdown of either JfNLRP3 or JfCaspase-1 reduces the serum JfIL-1β level, and promotes the bacterial colonization in systemic immune organs at 2 day-post infection, while overexpression of JfNLRP3 or JfCaspase-1 hampers the bacterial colonization in these organs of Japanese flounder. Taken together, our results identified the NLRP3 inflammasome paradigm in Japanese flounder, which not only providing new insight into the molecular mechanisms of teleost NLRP3 inflammasome and revealing its role in restricting bacterial infection in vivo, but also shedding light on the evolutionary of NLRP3 inflammasome in teleost.