Human telomerase RNA (hTR) is an intrinsic component of telomerase enzyme. Small interfering RNAs (siRNAs) and single-stranded antisense oligonucleotides have been used previously for silencing of the hTR. The objective of this study was to investigate the effect of partially double-stranded oligodeoxynucleotides (ODNs), in vitro and in vivo in comparison to single-stranded antisense ODNs and siRNAs. ODNs were designed on the basis of structural properties of an ODN from previous studies on HIV, to target the hTR in the human cervical carcinoma HeLa cell line and mouse telomerase RNA (mTR) in the murine metastatic melanoma B16-F10 cell line, respectively. Our results indicate that ODNs were able to inhibit the hTR by 68% and the mTR by 81% in the respective cell lines. This correlated with ODN-mediated rapid inhibition of cell proliferation and induction of apoptosis excluding slow effects on telomerase function. The inhibition of the hTR was decreased by knock-down of the cellular RNases H suggesting their contribution. Furthermore, we showed a reduction in numbers of metastases by 70% after intravenous administration of ODN-transfected B16-F10 cells in C57BL/6 mice. Our study demonstrates the potential utility of these hairpin-loop-structured ODNs as a different group of nucleic acids for telomerase-based antiproliferative strategies.