Mitochondria have a multitude of functions ranging from energy generation to regulation of cellular processes. Mitochondrial dynamics (the balance between mitochondrial fission and fusion) has been implicated in regulating key macrophage functions, such as inflammatory and antimicrobial responses. This study focused on molecular mechanisms responsible for Toll-like Receptor (TLR)-inducible mitochondrial fission in macrophages. Here we reveal that lipopolysaccharide (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, was also required for LPS-inducible fission; LPS-inducible fission was defective in both Drp1-/- mouse embryonic fibroblasts and Drp1-silenced BMM. Furthermore, LPS increased Drp1 recruitment to mitochondria, with acute LPS-induced Drp1 phosphorylation at serine 635 (S635) and delayed Drp1 dephosphorylation at serine 656 (S656) correlating with the early and late fission responses, respectively. Since fission has been linked to LPS-inducible glycolysis, the role of the glycolysis-promoting class IIa histone deacetylase (HDAC), Hdac7, in LPS-inducible fission was next investigated. Fission was abrogated in Hdac7-deficient macrophages and amplified in primary macrophages that overexpress Hdac7. Pre-treatment of BMM with the class IIa HDAC inhibitor TMP195 resulted in decreased LPS-inducible fission. However, both wild type Hdac7 and enzyme-dead Hdac7 could restore LPS-inducible fission in Hdac7-deficient BMM. This suggests that the deacetylase activity of Hdac7 is not required for Drp1-mediated fission in macrophages. Interestingly, LPS-mediated S656 dephosphorylation was abrogated in both MyD88- and Hdac7-deficient BMM, suggesting that this modification may be particularly important for TLR-inducible fission. In summary, this study has thus far delineated a pathway in which TLR4 activation promotes Drp1-mediated mitochondrial fission in a MyD88- and Hdac7-dependent pathway.