Significance Repetitive DNA sequences are abundant in the human genome, and their high variabilities contribute to genetic diversity and disease susceptibility. Here, we report that an intronic variable number tandem repeat element, VNTR2-1, is critical for the transcription of the human telomerase reverse transcriptase ( hTERT ) gene in a cell-context–dependent manner. Removal of this element at its native genomic site in cancer cells resulted in telomere shortening, cellular senescence, and impaired tumor growth. VNTR2-1 length, consisting of 53 to 160 copies of 42-bp repeats, varies widely in human populations. hTERT alleles with short VNTR2-1 are underrepresented in African American centenarians, suggesting that hTERT regulation by VNTR2-1 plays a role in human aging and tumorigenesis.