Background: Sepsis is a life-threatening organ dysfunction initiated by a dysregulated response to infection, with imbalanced inflammation and immune homeostasis. Macrophages play a pivotal role in sepsis. N-[1-(1-oxopropyl)-4-piperidinyl]-N'-[4-(trifluoromethoxy)phenyl)-urea (TPPU) is an inhibitor of soluble epoxide hydrolase (sEH), which can rapidly hydrolyze epoxyeicosatrienoic acids (EETs) to the bio-inactive dihydroxyeicosatrienoic acids. TPPU was linked with the regulation of macrophages and inflammation. Here, we hypothesized that sEH inhibitor TPPU ameliorates cecal ligation and puncture (CLP)-induced sepsis by regulating macrophage functions. Methods: A polymicrobial sepsis model induced by CLP was used in our study. C57BL/6 mice were divided into four groups: sham+ phosphate buffer saline (PBS), sham+TPPU, CLP+PBS, CLP+TPPU. Mice were observed 48 h after surgery to assess the survival rate. For other histological examinations, mice were sacrificed 6 h after surgery. Macrophage cell line RAW264.7 was used forin vitrostudies. Results: TPPU treatment, accompanied with increased EETs levels, markedly improved the survival of septic mice induced by CLP surgery, which was associated with alleviated organ damage and dysfunction triggered by systemic inflammatory response. Moreover, TPPU treatment significantly inhibited systemic inflammatory response via EETs-induced inactivation of mitogen-activated protein kinase signaling due to enhanced macrophage phagocytic ability and subsequently reduced bacterial proliferation and dissemination, and decreased inflammatory factors release. Conclusion: sEH inhibitor TPPU ameliorates cecal ligation and puncture-induced sepsis by regulating macrophage functions, including improved phagocytosis and reduced inflammatory response. Our data indicate that sEH inhibition has potential therapeutic effects on polymicrobial-induced sepsis.