Imbalance of macrophage polarization plays an indispensable role in acute lung injury (ALI), which is considered as a promising target. Matrix metalloproteinase-9 (MMP-9) is expressed in the macrophage, and has a pivotal role in secreting inflammatory cytokines. We reported that saquinavir (SQV), a first-generation human immunodeficiency virus-protease inhibitor, restricted exaggerated inflammatory response. However, whether MMP-9 could regulate macrophage polarization and inhibit by SQV is still unknown. We focused on the important role of macrophage polarization in CLP (cecal ligation puncture)-mediated ALI and determined the ability of SQV to maintain M2 over M1 phenotype partially through the inhibition of MMP-9. We also performed a limited clinical study to determine if MMP-9 is a biomarker of sepsis. Lipopolysaccharide (LPS) increased MMP-9 expression and recombinant MMP-9 (rMMP-9) exacerbated LPS-mediated M1 switching. Small interfering RNA to MMP-9 inhibited LPS-mediated M1 phenotype and SQV inhibition of this switching was reversed with rMMP-9, suggesting an important role for MMP-9 in mediating LPS-induced M1 phenotype. MMP-9 messenger RNA levels in peripheral blood mononuclear cells of these 14 patients correlated with their clinical assessment. There was a significant dose-dependent decrease in mortality and ALI after CLP with SQV. SQV significantly inhibited LPS-mediated M1 phenotype and increased M2 phenotype in cultured RAW 264.7 and primary murine bone marrow-derived macrophages as well as lung macrophages from CLP-treated mice. This study supports an important role for MMP-9 in macrophage phenotypic switching and suggests that SQV-mediated inhibition of MMP-9 may be involved in suppressing ALI during systemic sepsis.