Objective: To investigate the role of mitochondrial reactive oxygen species (mtROS) in NLRP3 inflammasome activation in cardiac fibroblast (CF). Methods: Primary mouse CF was cultured. CFs were divided into control group, initiation group (LPS), activation group (LPS+ATP) and intervention group (LPS+mito-TEMPO+ATP). MtROS in CF was detected with MitoSOX reagent. CF NLPR3 inflammasome activation was assessed with Western blotting for intracellular NLRP3, ASC, Pro-caspase-1,Pro-IL-1β and caspase-1 p20,IL-1β in the supernatant and ELISA was used to detect the level of IL-1β.The interaction between NLRP3 and ASC was examined by co-immunoprecipitation. Results: The expression of intracellular mtROS in the activation group was significant increased compared with the control group (P<0.05), while the mtROS expression in the intervention group was lower than that in the activation group (P<0.05). After challenging by LPS, the CF intracellular NLRP3 and Pro-IL-1β were significantly higher than the control group (P<0.05). Pro-IL-1β was higher in the intervention group than in the activation group (P<0.05). The caspase-1 p20 and IL-1β in the supernatant of the activation group were higher than those in the control group (P<0.05), the caspase-1 p20 and IL-1β in the intervention group were significantly lower than those in the activation group (P<0.05). Protein NLRP3 interacted with ASC in activation group, and the binding of NLRP3 and ASC in the intervention group was reduced. Conclusion: MtROS promoted the NLRP3 inflammasome activation in CF via facilitated the binding of NLRP3 and ASC.