Mitochondrial DNA (mtDNA) replication is essential for mitochondrial function in response to developmental and physiological demand. Impaired mtDNA replication leads to developmental defects, aging and diseases. To identify novel genes involved in the regulation of mtDNA replication, we conducted an in vivo RNAi screen in Drosophila and identified a novel gene, EmptyHouse. We found that the protein encoded by EmptyHouse is associated with the mitochondrial inner membrane. EmptyHouse mutant has severe developmental delay and is lethal during pupa and adult stages. Genetic analysis indicates that this gene has an essential role in mtDNA replication. Loss of this gene results in impaired mitochondrial oxidative phosphorylation activity. The human homolog, WBSCR16, is one of the genes that flanking the Williams-Beuren syndrome (WBS) commonly deleted region. The function of WBSCR16 in human is completely unknown. We expressed WBSCR16 in human tissue culture cells and found it expressed in mitochondria. Knocking down WBSCR16 expression in human tissue culture significantly slows down the mtDNA replication rate. Further characterization of EmptyHouse mutant and its human homolog may provide insight into the mechanism of regulation of mtDNA replication during development and the pathogenesis of diseases.