NLRX1 weakens lipopolysaccharide (LPS)-induced NF-kappa B activation on immune cells. Cytochrome P450 epoxygenase 2J2 (CYP2J2) attenuates LPS-induced cardiac injury by inhibiting NF-kappa B activation. However, it is still unclear whether NLRX1 could reduce LPS-induced heart damage and whether it is involved in the anti-LPS cardioprotective effect of CYP2J2. In this study, we found that NLRX1 knockout further exacerbated LPS-induced heart injury and up-regulated the proinflammatory cytokines in serum and heart tissue, and weakened the inhibitory effect of CYP2J2 on the harmful effects caused by LPS. We also found that LPS treatment induced ubiquitination of NLRX1 and promoted its binding to IKK alpha/beta in myocardial tissue, which should theoretically inhibit NF-kappa B activation. However, LPS eventually leads to activation of NF-kappa B and NLRP3 inflammasome. Under the action of LPS, CYP2J2 further promoted the ubiquitination of NLRX1 and its binding to IKK alpha/beta, impaired NF-kappa B activation and NLRP3 inflammasome activation. NLRX1 knockout notably aggravated LPS-induced NF-kappa B activation and NLRP3 inflammasome activation, and attenuated the inhibitory effects of CYP2J2 on NF-kappa B signal and NLRP3 inflammasome. More, CYP2J2 reduced LPS-induced reactive oxygen species (ROS) production and mitochondrial depolarization in heart cells, thereby inhibiting NLRP3 inflammasome activation. NLRX1 knockdown aggravated mitochondrial depolarization induced by LPS and weakened the protective effect of CYP2J2 on mitochondrial potential, although it had no significant effect on reactive oxygen species production. Together, these findings demonstrated that NLRX1 knockout aggravated LPS-induced heart injury and weakened the anti-LPS cardioprotective effect of CYP2J2 by enhancing activation of NF-kappa B and NLRP3 inflammasome.