Bacterial infection and its associated sepsis are devastating clinical entities that lead to high mortality and morbidity in critically ill patients. Phagocytosis, along with other innate immune responses, exerts crucial impacts on the outcomes of these patients. MicroRNAs (miRNAs) are a novel class of regulatory noncoding RNAs that target specific mRNAs for modulation of translation and expression of a targeted protein. The roles of miRNAs in host defense against bacterial sepsis remain unclear. We found that bacterial infections and/or bacterial-derived LPS enhanced the level of miR-15a/16 in bone marrow-derived macrophages (BMDMs). Deletion of miR-15a/16 (miR-15a/16(-/-)) in myeloid cells significantly decreased the bacterial infection-associated mortality in sepsis mouse models. Moreover, miR-15a/16 deficiency (miR-15a/16(-/-)) resulted in augmented phagocytosis and generation of mitochondrial reactive oxygen species in BMDMs. Supportively, overexpression of miR-15a/16 using miRNA mimics led to decreased phagocytosis and decreased generation of mitochondrial reactive oxygen species. Mechanistically, deletion of miR-15a/16 upregulated the expression of TLR4 via targeting the principle transcriptional regulator PU.1 locating on the promoter region of TLR4, and further modulated the downstream signaling molecules of TLR4, including Rho GTPase Cdc 42 and TRAF6. In addition, deficiency of miR-15a/16 also facilitated TLR4-mediated proinflammatory cytokine/chemokine release from BMDMs at the initial phase of infections. Taken together, miR-15a/16 altered phagocytosis and bacterial clearance by targeting, at least partially, on the TLR4-associated pathways, subsequently affecting the survival of septic mice.