Abstract Purpose Determine the efficacy and timing of small molecule oligonucleotide-based inhibitors to the enzyme telomerase in an in vitro model of androgen-independent, osseous prostate cancer. Materials and Methods Telomerase was inhibited in prostate cancer cell lines C4-2/C4–2B and in controls by using small molecule antisense oligonucleotide-based inhibitors alone or in various combinations of small-dose Taxotere® (sanofi-aventis, Bridgewater, NJ) and/or conditionally replication competent adenovirus (AD-BSP-E1a). After transfection and proliferation, telomerase telomeric repeat amplification protocol and telomere restriction fragment assays were performed, with specific times for evaluating telomere length. Specimens were stained for analysis with hematoxylin and eosin (H&E), terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL), and prostate-specific antigen (PSA). Results C4-2/C4–2B cell lines had the shortest initial mean telomere length (approximately 2.5 kilobase [kb]) compared to PC-3 (approximately 5.5 kb). Dose-dependent inhibition of telomerase activity was seen using match oligonucleotide-based inhibitors to telomerase (50% inhibitory concentration 3–5 nm), whereas mismatch compound showed no telomerase inhibition. Significant growth delay and apoptosis in cell lines occurred after >50 days of treatment. Cells treated with combination “triple therapy” (i.e., telomerase inhibitors, adenovirus, and Taxotere®) had the highest amount of apoptosis. Compared to controls, all combination treatment groups had statistically significant reductions in prostate-specific antigen in the conditioned media. Conclusions Combining cytotoxic regimens with small molecule inhibitors to telomerase with oligonucleotide-based agents could be beneficial in controlling osseous hormone refractory prostate cancer, as evidenced by these in vitro, preclinical investigations. Telomerase inhibition needs to move into in vivo models and human studies.