The invention relates to a mitochondrial expression vector which may comprise a gene to be expressed and/or a selection marker and a mitochondrial region, and a method for inserting a DNA to be expressed into mitochondria of mammalian cells, wherein the method may comprise the steps: (i) construction of a mitochondrial expression vector or a mitochondrial genome, (ii) reversible induction of megamitochondria and (iii) transfection of the megamitochondria by means of a physical transfection method.