In eukaryotes, cytosine methylation of nuclear DNA at CpG sequences (CpG) regulates epigenetic inheritance through alterations in chromatin structure. However, mitochondria lack nucleosomal chromatin, therefore the molecular mechanisms by which CpG influences mitochondria must be different and are as yet unknown. Mitochondrial Transcription Factor A (TFAM) is both the primary DNA-compacting protein in the mitochondrial DNA (mtDNA) nucleoid and a transcription-initiation factor. TFAM must encounter hundreds of CpGs in mtDNA, so the occurrence of CpG has the potential to impact TFAM-DNA recognition. We used biophysical approaches to determine whether CpG alters any TFAM-dependent activities. CpG in the heavy strand promoter (HSP1) increased the binding affinity of TFAM and induced TFAM multimerization with increased cooperativity compared to nonmethylated DNA. However, CpG had no apparent effect on TFAM-dependent DNA compaction. Additionally, CpG had a clear and context-dependent effect on transcription initiating from the three mitochondrial promoters. Taken together, our findings demonstrate that CpG in the mitochondrial promoter region does impact TFAM-dependent activities in vitro.