Objective To construct the retroviral vector carrying novel gene mgt-16 and to investigate its expression in mouse mesenchymal stem cells C3H/10T1/2(10T1/2). Methods DNA sequences containing mouse novel gene mgt-16 was used as a template for PCR amplification of full length mgt-16 cDNA. Then the DNA fragment was cloned into pEGFP-N1 vector to produce pEGFP-N1-16 vector after T-A cloning with pMD18T plasmid and sequencing. The pEGFP-N1-16 vector was confirmed by PCR, restriction enzyme digestion and sequencing analysis. The retroviral vector, pLEGFP-N1-16, was constructed using retroviral vector, pLEGFP-N1, and pEGFP-N1-16 vector including mgt-16 gene. The pLEGFP-N1-16 vector was verified by restriction enzyme digestion, sequenced, and then transfected into packaging cell line Phoenix to prepare EGFP fused mgt-16 retrovirus particles, which were collected and used to infect 10T1/2 cells. G418 (400 μg/mL) continuous selection was conducted to obtain 10T1/2 cell clones stably overexpressing EGFP fused mgt-16. Fluorescence microscope was employed to determine the expression and subcellular localization of MGT-16 in Phoenix and 10T1/2 cells. Results A band of about 300 bp size was observed by agarose gel electrophoresis after PCR amplification for mgt-16 gene, and the result of sequencing showed that the sequence of insert fragment in T-A clones was identical to mgt-16 gene reported in Genbank. PCR, restriction enzyme digestion and sequencing revealed that the pEGFP-N1-16 plasmid was successfully constructed. Restriction enzyme digestion and sequencing revealed that the pLEGFP-N1-16 plasmid was also successfully constructed. Phoenix and 10T1/2 cells overexpressing MGT-16 showed green fluorescence distribution in the cytoplasmic, especially around the perinuclear area. Conclusion We have successfully constructed a recombinant retroviral vector carrying novel gene, mgt-16, and expressed it in mouse mesenchymal stem cells, which provides a basis for studying the role of novel gene mgt-16 in mesenchymal stem cells.