Bacterial toxin DddA-derived cytosine base editors (DdCBEs)-composed of split DddA (a cytosine deaminase specific to double-stranded DNA), custom-designed TALE (transcription activator-like effector) DNA-binding proteins, and a uracil glycosylase inhibitor-enable mitochondrial DNA (mtDNA) editing in human cells, which may pave the way for therapeutic correction of pathogenic mtDNA mutations in patients. The utility of DdCBEs has been limited by off-target activity, which is probably caused by spontaneous assembly of the split DddA deaminase enzyme, independent of DNA-binding interactions. We engineered high-fidelity DddA-derived cytosine base editors (HiFi-DdCBEs) with minimal off-target activity by substituting alanine for amino acid residues at the interface between the split DddA halves. The resulting domains cannot form a functional deaminase without binding of their linked TALE proteins at adjacent sites on DNA. Whole mitochondrial genome sequencing shows that, unlike conventional DdCBEs, which induce hundreds of unwanted off-target C-to-T conversions in human mtDNA, HiFi-DdCBEs are highly efficient and precise, avoiding collateral off-target mutations, and as such, they will probably be desirable for therapeutic applications.