Spermidine/spermine N1‐acetyltransferase (SSAT) is the rate‐limiting step in polyamine catabolism. In a previous study, we constructed a recombinant adenovirus, Ad‐SSAT, which can express human SSAT. In the present study, we investigated the effect of upregulated and downregulated SSAT on gastric cancer cells. We found that upregulated SSAT could inhibit the growth of MGC803 and SGC7901 cells, whereas adverse results were found with downregulated SSAT. We further analyzed cell cycle profiles and the expression levels of the major cell cycle regulatory proteins of S phase. The results showed that the growth inhibition was caused by S phase arrest. Ad‐SSAT suppressed the expression of cyclin A and nuclear factor E2F1 in MGC803 and SGC7901 cells. We observed the E2F promoter activity caused by Ad‐SSAT using a reporter gene assay. We also investigated the antitumorigenicity of upregulated SSAT by Ad‐SSAT using a SGC7901 xenograft model in nude mice. Our results suggest that the upregulation of SSAT by Ad‐SSAT infection inhibited the growth of gastric cancer in vitro and in vivo. Ad‐SSAT arrested gastric cancer cells in S phase, which was mediated through downregulation of the cyclin A‐E2F signaling pathway. (Cancer Sci 2009); 000: 000–000