Mitochondria have a multitude of functions ranging from energy generation to regulation of cellular processes. This study focuses on a novel mitochondrial function relating to control of macrophage antimicrobial responses. Recent evidence suggests that mitochondrial dynamics (the balance between mitochondrial fission and fusion) regulates key macrophage functions, such as phagocytosis. Here we reveal that LPS stimulation of mouse bone marrow-derived macrophages (BMM) promotes toll-like receptor 4 (Tlr4)-dependent mitochondrial fission. Fission is triggered as early as 1 h post-stimulation, is sustained over a 24 h time course, and is dependent on the TLR adaptor protein MyD88. Drp1, a GTPase that is essential for mitochondrial fission in other cellular systems, was also required for LPS-inducible fission; the LPS-inducible fission response was defective in both Drp1-/- mouse embryonic fibroblasts, as well as Drp1-silenced BMM. Furthermore, acute LPS-induced Drp1 phosphorylation at serine 635 (S635) correlated with acutely inducible fission, whereas Drp1 dephosphorylation at serine 656 (S656) coincided with the late sustained fission response. LPS-induced S656 dephosphorylation was abrogated in MyD88-deficient BMM, suggesting that this post-translational modification may be particularly important for TLR-inducible fission. In investigating biological functions of this pathway, we found that pharmacological or genetic targeting of TLR-inducible fission compromised the ability of macrophages to clear a non-pathogenic E. coli K-12 strain. Further investigation of the potential antimicrobial mechanisms identified a likely role for inducible gene expression, rather than mitochondrial ROS, in bacterial clearance. This study has thus far delineated a pathway in which TLR4 activation promotes Drp1-dependent mitochondrial fission to trigger macrophage antimicrobial responses. Given their ancestry, we speculate that mitochondrial division may mimic intracellular bacterial replication to alert the innate immune system of a bacterial threat.