A novel antisense transcript was identified in the human telomerase reverse transcriptase (hTERT) promoter region, suggesting that the hTERT promoter is bidirectional. This transcript, named hTERT antisense promoter-associated (hTAPAS) RNA, is a 1.6 kb long non-coding RNA. hTAPAS transcription is initiated 167 nucleotides upstream of the hTERT transcription start site and is present in both the nucleus and the cytoplasm. Surprisingly, we observed that a large fraction of the hTERT polyadenylated RNA is localized in the nucleus, suggesting this might be an additional means of regulating the cellular abundance of hTERT protein. Both hTAPAS and hTERT are expressed in immortalized B-cells and human embryonic stem cells but are not detected in normal somatic cells. hTAPAS expression inversely correlates with hTERT expression in different types of cancer samples. Moreover, hTAPAS expression is not promoted by an hTERT promoter mutation (-124 C>T). Antisense-oligonucleotide mediated knockdown of hTAPAS results in an increase in hTERT expression. Conversely, ectopic overexpression of hTAPAS down regulates hTERT expression, suggesting a negative role in hTERT gene regulation. These observations provide insights into hTAPAS as a novel player that negatively regulates hTERT expression and may be involved in telomere length homeostasis.