Objective To construct the recombinant eukaryotic expression vector of human mitochondrial transcription termination factor 2 (MTERF2) gene and determine the cellular localization by overexpressing MTERF2 in human Caski cervical cancer cells. Methods The coding sequence of MTERF2 was amplified by reverse transcription-PCR using the total RNA extracted from human cervical cancer Caski cells, and then was inserted into p3×FLAG-CMV-14 vector. After being verified by PCR and DNA sequencing, the positive recombinant plasmid was transiently transfected into Caski cells. Western blotting and immunofluorescence technique were performed to analyze the expression and distribution of human MTERF2 proteinat 24, 32 and 48 hours after transfection. Results Sequence analysis showed that the correct target gene (1158 bp) was inserted into the vector at the expected position. The target protein band was detected at Mr 44 000 as we had predicted in the transfected cells while not in the negative control group, which indicated MTERF2 expression vector could be successfully transfected and expressed in Caski cells. The p3×FLAG-MTERF2 protein was highly expressed and displayed a mitochondrial distribution at 24 hours post-transfection in Caski cells. Conclusion We successfully constructed the eukaryotic expression plasmid p3×FLAG-CMV-MTERF2 and expressed p3×FLAG tagged MTERF2 effectively in the mitochondria of Caski cells.