Mitochondrial dynamics including mitochondrial fusion is implicated in innate immune responses. Here we demonstrate that absence of the mitochondrial fusion protein mitofusin1 (MFN1) enhanced NLRP3 inflammasome-mediated caspase-1 activation and the subsequent secretion of interleukin-1beta (IL-1β) and IL-18 in macrophages. Absence of MFN1 increased the levels of mitochondrial DNA (mtDNA) mutation, but not mtDNA copy number. We further observed that NLRP3 inflammasome activation was enhanced in macrophages from mtDNA mutator mice expressing proofreading-deficient mtDNA polymerase gamma. Both mtDNA mutator mice and myeloid cell-specific Mfn1 deficient mice displayed increased systemic IL-1β production and mortality in a cecal ligation and puncture (CLP) -induced sepsis. In consistence with our findings from murine models, the levels of mitochondrial heteroplasmy, the co-existence of wild type and mutated mtDNA, in human white blood cells were significantly associated with disease severity of critically ill patients and increased in patients with sepsis. Collectively, our study demonstrates that MFN1 regulates NLRP3 inflammasome-dependent inflammation in sepsis by preventing the accumulation of mtDNA mutation.