Telomerase is proposed as an anticancer target. Increasing evidence suggests that telomerase is involved in functions independent of telomere-extension activity. In this study, we designed a small interfering RNA (siRNA) targeting human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase. Using transient and persistent transfection of hTERT siRNA into telomerase-positive human colon carcinoma HCT116 cells, we demonstrated that hTERT siRNA suppresses hTERT expression and leads to inhibition of telomerase activity and HCT116 cell growth and in vivo tumorigenicity in nude mice. Further analysis indicated that depletion of hTERT reduces cell adhesion, migration, and invasion prior to inhibition of cell proliferation. Downregulation of hTERT also decreased the expression levels of adhesion- and motility-related proteins, in particular c-Met and integrins. The lack of detectable changes in telomere length showed that downregulation of hTERT in the present system had no significant effect on telomere-extension activity. Taken together, our results highlight the therapeutic potential of a novel hTERT siRNA and suggest that hTERT is involved in the regulation of cell motility in a telomere-independent manner.