Sepsis is a systemic inflammatory condition associated with severe organ failure, particularly splenic injury. Fraxetin (Fra), a natural product isolated from ash bark, exhibits anti-inflammatory and antioxidant properties. This study explores the function and mechanism of Fra in sepsis-induced splenic injury using an in vivo mouse model induced by Cecal Ligation and Puncture and an in vitro sepsis model based on LPS/ATP co-stimulated J774A.1 cells. The experimental groups are as follows: Sham operation or control group, Fra control group, CLP or LPS/ATP group, CLP + Fra group or LPS/ATP + Fra group, with Dexamethasone as a positive control. The results indicated that Fra improved the survival rate, inhibited bacteria burden, and reduced spleen edema. Fra also alleviated spleen necrosis, and restored the structural integrity. Blood results showed that Fra restored platelet count and lymphocyte percentage, reduced neutrophil ratio and C-reactive protein increase, and prevented lymphocyte depletion. Immunohistochemistry demonstrated that Fra inhibited MPO levels. Additionally, Fra downregulated Procalcitonin, inhibited pro-inflammatory cytokines, NO release and Arg-1 expression, illustrating its anti-inflammatory effects. DHE staining revealed that Fra inhibited ROS and MDA, enhanced CAT, GSH-PX, and SOD activities. Furthermore, Fra inhibited NLRP3 inflammasome activation, p-IKKβ expression and NF-κB pathway. Mechanistically, molecular docking studies revealed that Fra could bind to IKKβ, thereby blocking the NF-κB pathway and NLRP3 inflammasome, functioning anti-inflammatory effects. In summary, Fra targets IKKβ to block the NF-κB pathway and NLRP3 inflammasome activation, alleviating sepsis-induced splenic injury, making it a promising therapeutic strategy for treating sepsis-induced splenic injury.