The human telomerase reverse transcriptase (hTERT) gene has been used to stimulate the proliferation of most types of human cells. The present study was designed to evaluate the feasibility and efficiency of adenovirus-mediated hTERT in the proliferation of bovine mammary gland epithelial cells (bMGEs). A plasmid and an adenovirus vector that carried hTERT, namely pEGFP- hTERT and Ad- hTERT, were constructed and transfected into bMGEs, respectively. In order to select the best strategy for stimulating cell proliferation, the adenovirus- and plasmid-mediated hTERT were compared in terms of the positive cloning and transgenic efficiency. The results showed that only Ad- hTERT had high infection efficiency and produced a positive polyclone population (hTERT-bMGEs). The characteristics of the hTERT-bMGEs were investigated with further analysis by reverse transcription-polymerase chain reaction (RT-PCR), western blotting, proliferation assays, and flow cytometry, which showed that hTERT facilitated strong cell proliferation. Real-time quantitative PCR showed a normal level of expression of beta-casein, the caspase-8 and c-myc proto-oncogene, and immunofluorescence demonstrated the properties of the epithelial cells. In conclusion, the adenovirus-mediated hTERT gene could not only extend the cell lifespan, but also maintained the primary characteristics of the cells. It may be possible to extend the use of a wide variety of non-human mammalian cells in this way. This study has provided additional insight into the mechanism of cell proliferation by demonstrating the lack of integration of the adenovirus-mediated hTERT gene into the mammalian genome.