Previous in vitro and in vivo studies have suggested that lactobacilli can exert antiproliferative effects on the gastrointestinal epithelium. However, their role in affecting the cellular proliferative mechanisms is not completely clear. The aim of this study was to investigate the effects of increasing concentrations of Lactobacillus rhamnosus strain GG (L. GG) homogenate on cell growth and proliferation (by MTT, [3H]-thymidine incorporation and polyamine biosynthesis) in neoplasms originating from different gastrointestinal tracts. Thus, HGC-27 human gastric cancer cells and DLD-1 human colonic adenocarcinoma cells were evaluated. Besides, in order to verify which bacterial fraction was involved in the antiproliferative effects, the cytoplasm and cell wall extracts were tested separately. Gastric HGC-27 and colonic DLD-1 cells showed significant differences in their proliferative behavior, in particular in their polyamine profile and biosynthesis. Notwithstanding, one and the other proved to be sensitive to the growth inhibition by the highest concentrations of bacterial homogenate. Both HGC-27 and DLD-1 cells were resistant to the bacterial cell wall fractions, whereas increasing cytoplasm fraction concentrations induced an evident antiproliferative effect. These data suggest that cytoplasm extracts could be the responsible for L. GG action on proliferation in these two cell lines from gastric and colonic neoplasms.