Telomerase activity is detected in more than 90% of examined tumors but not in most normal somatic cells. Among three subunits of human telomerase, human telomerase reverse transcriptase (hTERT) is the rate-limiting component for telomerase activity. Therefore, targeting hTERT represents a promising approach for diminishing telomerase function that will probably not cause substantial side effects on telomerase negative somatic cells. To explore the effects of antisense hTERT (ahTERT) on the malignant phenotypes of human SGC-7901 gastric cancer cell line in vitro, an antisense eukaryotic expression vector of hTERT was constructed by gene recombinant technology. Telomerase activity by telomeric repeat amplification protocol–ELISA, mRNA of telomerase subunits, c-myc and bcl-2 by reverse transcript-PCR, terminal restriction fragment (TRF) by Southern blot, cell cycle distribution by flow cytometry and protein expression of hTERT, c-myc and bcl-2 by Western blot were analyzed in SGC-7901 cells before and after transfection. Cloning efficiency assay in soft agar and tumorigenesis in nude mice were also examined and evaluated in the above cells. The results demonstrated that after ahTERT transfection, the proliferation of SGC-7901 cells was significantly inhibited. Further study showed that telomerase activity, telomere length, the mRNA and protein expression of hTERT, bcl-2 and c-myc were decreased in ahTERT-transfected cells. There were, however, no obvious effects on transcription of human telomerase RNA (hTR) and human telomerase associated protein1 (TP1) in both transfected and untransfected cells. Flow cytometric analysis displayed an accumulation of G0/G1 phase and a decreasing proliferation index (PI) in ahTERT-transfected cells. Moreover, no tumorigenicity was found after subcutaneous injection of ahTERT-transfected cells in nude mice, whereas palpable tumors were observed in mice injected with control cells. Our study indicates that exogenous ahTERT can inhibit proliferation and partially reverse malignant phenotypes of SGC-7901 cells via the suppression of telomerase activity, hTERT, c-myc and bcl-2 expression. Antisense technology targeted hTERT strategy might be a potential approach for gastric cancer therapy.