Rationale:Timely inhibition of inflammation and initiation of resolution are important to repair injured tissues. MST1/2 (mammalian STE20-like protein kinase 1/2) acts as a regulator of macrophage-associated immune responses to bacterial infections. However, the role of MST1/2 in regulating macrophage phenotype and function in myocardial infarction (MI) remains unclear.Objective:To determine the function and underlying mechanism of macrophage MST1/2 in cardiac repair post-MI.Methods and Results:UsingLysMCre-mediatedMst1/2-deficient mice, we found that MST1 deficiency exacerbated cardiac dysfunction after MI. Single-cell RNA sequencing assay indicated that the effect was attributed to a shift of macrophage subtypes from those expressingCxcl2andCd163towardCcl2andCcl4expression. Mass spectrometry identified LTB4 (leukotriene B4) as the lipid mediator that was upregulated in the absence of MST1. We found that MST1 phosphorylated 5-LOX (5-lipoxygenase) at its T218 residue, disrupting the interaction between 5-LOX and 5-LOX-activating protein, resulting in a reduction of LTB4 production. In contrast, a 5-LOXT218Avariant showed no response to MST1. Moreover, treatment of peritoneal macrophages with LTB4 or medium conditioned byMst1-deficient macrophages resulted in highCcl2andCcl4expression and lowCxcl2andCd163expression, except when the cells were co-treated with the BLT1 (LTB4 receptor 1) antagonist CP105696. Furthermore, CP105696 ameliorated cardiac dysfunction inLysMCre-mediatedMst1/2-deficient mice and enhanced cardiac repair in wild-type mice treated with XMU-MP-1 (4-((5,10-dimethyl-6-oxo-6,10-dihydro-5H-pyrimido[5,4-b]thieno[3,2-e][1,4]diazepin-2-yl)amino)benzenesulfonamide) after MI.Conclusions:Taken together, our results demonstrate that inhibition of MST1/2 impaired post-MI repair through activating macrophage 5-LOX–LTB4–BLT1 axis.