Mechanisms of mitochondrial DNA (mtDNA) maintenance have recently gained wide interest owing to their role in inherited diseases as well as in aging. Twinkle is a new mitochondrial 5'-3' DNA helicase, defects of which we have previously shown to underlie a mitochondrial disease, progressive external ophthalmoplegia with multiple mtDNA deletions. Mouse Twinkle is highly similar to the human counterpart, suggesting conserved function. Here, we have characterized the mouse Twinkle gene and expression profile and report that the expression patterns are not conserved between human and mouse, but are synchronized with the adjacent gene MrpL43, suggesting a shared promoter. To elucidate the in vivo role of Twinkle in mtDNA maintenance, we generated two transgenic mouse lines overexpressing wild-type Twinkle. We could demonstrate for the first time that increased expression of Twinkle in muscle and heart increases mtDNA copy number up to 3-fold higher than controls, more than any other factor reported to date. Additionally, we utilized cultured human cells and observed that reduced expression of Twinkle by RNA interference mediated a rapid drop in mtDNA copy number, further supporting the in vivo results. These data demonstrate that Twinkle helicase is essential for mtDNA maintenance, and that it may be a key regulator of mtDNA copy number in mammals.