Human telomerase reverse transcriptase (hTERT) is upregulated in most cancer cell types as well in immortalized cells. The underlying mechanism for such upregulation, however, remains largely unknown. We report here that bile acids under acidified media increase hTERT expression via c-myc activation in primary human gastric cancer cell lines. Human gastric cancer MKN28, MGC803 and SGC7901 cells were treated with 100 µM deoxycholic acid (DCA) or chenodeoxycholic acid (CDCA) with or without acidified media in the presence or absence of the c-myc inhibitor 10058-F4 for 24 h. hTERT and c-myc protein levels were determined by western blot analysis. hTERT and c-myc mRNA levels were determined by RT-PCR. The promoter activities of hTERT and c-myc transcription were determined using promoter reporter luciferase assays for both. Telomerase enzyme activity was analyzed by stretch PCR. hTERT mRNA and protein levels were significantly increased by bile acids in acidified media and were accompanied with enhanced telomerase activity. No changes were found at a pH of 7.0 or with acidified media alone. Similarly, the mRNA and protein levels of c-myc were also increased by bile acids in acidified media but not at a pH of 7.0 or with acidified media alone. Importantly, pharmacologic inhibition of c-myc using 10058-F4 prevented hTERT induction by DCA or CDCA in gastric cancer cells under acidic conditions. Bile acids (DCA and CDCA) under acidic conditions increased hTERT expression in human gastric cancer cells by activation of c-myc transcription. This suggests that acidified bile acids may promote tumorigenesis and affect cell ageing via telomerase activation.