In vivo expression of human telomerase is significantly different from that of mouse telomerase. To assess the basis for this difference, a bacterial artificial chromosome clone containing the entire hTERT (human telomerase reverse transcriptase) gene was introduced in mice. In these transgenic mice, expression of the hTERT transgene was similar to that of endogenous hTERT in humans, rather than endogenous mTERT (mouse telomerase reverse transcriptase). In tissues and cells showing a striking difference in expression levels between hTERT in humans and mTERT in mice (i.e., liver, kidney, lung, uterus, and fibroblasts), expression of the hTERT transgene in transgenic mice was repressed, mimicking hTERT in humans. The transcriptional activity of the hTERT promoter was much lower than that of the mTERT promoter in mouse embryonic fibroblasts or human fibroblasts. Mutational analysis of the hTERT and mTERT promoters revealed that a nonconserved GC-box within the hTERT promoter was responsible for the human-specific repression. These results reveal that a difference in cis-regulation of transcription, rather than transacting transcription factors, is critical to species differences in tissue-specific TERT expression. Our data also suggest that the GC-box-mediated, human-specific mechanism for TERT repression is impaired in human cancers. This study represents a detailed characterization of the functional difference in a gene promoter of mice versus humans and provides not only important insight into species-specific regulation of telomerase and telomeres but also an experimental basis for generating mice humanized for telomerase enzyme and its pattern of expression.