Mitochondrial transcription termination factor 2 (mTERF2) is a mitochondrial matrix protein that binds to the mitochondrial DNA. Previous studies have shown that overexpression of mTERF2 can inhibit cell proliferation, but the mechanism has not been well defined so far. This study aimed to present the binding pattern of mTERF2 to the mitochondrial DNA (mtDNA) in vivo, and investigated the biological function of mTERF2 on the replication of mtDNA, mRNA transcription, and protein translation. The mTERF2 binding to entire mtDNA was identified via the chromatin immunoprecipitation analysis. The mtDNA replication efficiency and expression levels of mitochondria genes were significantly inhibited when the mTERF2 was overexpressed in HeLa cells. The inhibition level of mtDNA content was the same with the decreased levels of mRNA and mitochondrial protein expression. Overall, the mTERF2 might be a cell growth inhibitor based on its negative effect on mtDNA replication, which eventually down-regulated all of the oxidative phosphorylation components in the mitochondria that were essential for the cell's energy metabolism.