Human mesenchymal stem cell (MSC) extracellular vesicles (EV) can reduce the severity of bacterial pneumonia, but little is known about the mechanisms underlying their antimicrobial activity. In the current study, we found that bacterial clearance induced by MSC EV in Escherichia coli pneumonia in C57BL/6 mice was associated with high levels of leukotriene (LT) B₄ in the injured alveolus. More importantly, the antimicrobial effect of MSC EV was abrogated by cotreatment with a LTB₄ BLT1 antagonist. To determine the role of MSC EV on LT metabolism, we measured the effect of MSC EV on a known ATP-binding cassette transporter, multidrug resistance-associated protein 1 (MRP1), and found that MSC EV suppressed MRP1 mRNA, protein, and pump function in LPS-stimulated Raw264.7 cells in vitro. The synthesis of LTB₄ and LTC₄ from LTA₄ are competitive, and MRP1 is the efflux pump for LTC₄ Inhibition of MRP1 will increase LTB₄ production. In addition, administration of a nonspecific MRP1 inhibitor (MK-571) reduced LTC₄ and subsequently increased LTB₄ levels in C57BL/6 mice with acute lung injury, increasing overall antimicrobial activity. We previously found that the biological effects of MSC EV were through the transfer of its content, such as mRNA, microRNA, and proteins, to target cells. In the current study, miR-145 knockdown abolished the effect of MSC EV on the inhibition of MRP1 in vitro and the antimicrobial effect in vivo. In summary, MSC EV suppressed MRP1 activity through transfer of miR-145, thereby resulting in enhanced LTB₄ production and antimicrobial activity through LTB₄/BLT1 signaling.