Chemoprevention by dietary constituents has recently emerged as a novel approach to control gastric cancer incidence. Over the past years, functional foods and food supplements, especially probiotics, have received much attention as potential dietary cancer prevention agents. The precise mechanisms by which these lactic cultures exert their antitumorigenic activities are not fully elucidated, but there is some evidence of their influence on cell proliferation and growth. Ornithine decarboxylase (ODC) and spermidine/spermine N1-acetyltransferase (SSAT) are the key enzymes involved in polyamine biosynthesis and catabolism, respectively. These polycationic compounds are significantly associated with cancer risk and represent a specific markers for neoplastic proliferation. The aim of this study was to investigate the effects of increasing concentrations of Lactobacillus rhamnosus strain GG (ATCC 53103) (L. GG) homogenate on polyamine biosynthesis and polyamine degradation as well as on resulting polyamine levels in HGC-27 human gastric cancer cells. The influence of this probiotic on cell proliferation was also evaluated. Administration of probiotic homogenate significantly reduced both ODC mRNA and activity as well as polyamine content and neoplastic proliferation. Besides, an increase in both SSAT mRNA and activity occurred after LGG administration in HGC-27. These data suggest that a nutritional component such as the probiotic L. GG could be proposed in an alternative approach to prevention of gastric cancer. This strategy could overcome the limitations due to a prolonged use of drugs and/or the occurrence of their adverse effects, and it could reasonably also start at a young age.