Pot1, a telomere end-binding protein in fission yeast and human, is proposed not only to cap telomeres but also to recruit telomerase to the ends of chromosomes. No study has been performed regarding Pot1 expression status in human cancers. Thus, we examined POT1 mRNA expression in 51 gastric cancer (GC) tissues and evaluated telomere length and 3' telomeric overhang signals in 20 of the 51 GC tissues. Quantitative reverse transcription-PCR analysis showed that POT1 expression levels in the tumor relative to those in nonneoplastic mucosa (T/N ratio) were significantly higher in stage III/IV tumors than in stage I/II tumors (P = 0.005). Down-regulation of POT1 (T/n < 0.5) was observed more frequently in stage I/II GC (52.4%, 11 of 21) than in stage III/IV GC (23.3%, 7 of 30; P = 0.033), whereas up-regulation of POT1 (T/n > 2.0) was observed more frequently in stage III/IV GC (33.3%, 10 of 30) than in stage I/II GC (9.5%, 2 of 21; P = 0.048). POT1 expression levels showed decreased in accordance with telomere shortening (r = 0.713, P = 0.002). In-gel hybridization analysis showed that 3' telomeric overhang signals decreased in accordance with decreases in POT1 expression levels (r = 0.696, P = 0.002) and telomere shortening (r = 0.570, P = 0.013). Reduced POT1 expression was observed in GC cell lines with telomeres shortened by treatment with azidothymidine. In addition, inhibition of Pot1 by antisense oligonucleotides led to telomere shortening as well as inhibition of telomerase activity in GC cells. Moreover, inhibition of Pot1 decreased 3' overhang signals and increased the frequency of anaphase bridge (P = 0.0005). These data suggest that Pot1 may play an important role in regulation of telomere length and that inhibition of Pot1 may induce telomere dysfunction. Moreover, changes in POT1 expression levels may be associated with stomach carcinogenesis and GC progression.