Human complex I is the most intricate membrane-bound enzyme known to date, and the dysfunction of this enzyme complex, especially the mitochondrially encoded subunits such as ND4L, has linked to a wide variety of mitochondrial diseases, including Leber Hereditary Optic Neuropathy (LHON). Unfortunately, there is no specific and efficient therapy for treatment of diseases resulting from defects in the mitochondrial genome. Allotopic expression is a newly developed approach which has been considered to have the potential in conquering this challenging task. In this method, a mitochondrial gene is reengineered and expressed in the cytoplasm, and the resultant gene product is then imported back to mitochondria by the addition of mitochondrial targeting sequences (MTSs). In this study, we tested the possibility of applying this approach for human complex I ND4L subunit. We successfully designed and synthesized two transgenes, COX8MTSND4L and COX4MTSND4L, and used them in a tetracycline–inducible system for allotopic expression of ND4L by transient transfection. We demonstrated that the recoded ND4L transgene could be expressed by the addition of tetracycline, and this inducible system was also applicable to a nuclear-encoded mitochondrial gene for NDUFV2 (24k) subunit of human complex I. However, the two mitochondrial targeting sequences, COX8MTS and COX4MTS, we chose were unable to carry our targeted ND4L to the correct location in the mitochondria. The highly hydrophobic property of the ND4L may impede its mitochondrial import. We suggest that using other different MTSs, adopting 3’ untranslated region (3’ UTR) in the targeted transgene, looking for proteins which can stabilize the mRNAs encoding mitochondrial proteins or any combination of these three methods may improve the applicability of allotopic expression of ND4L.