The aim of this study was to investigate the effect of outer membrane vesicle (OMV) of Salmonella enterica serovar Typhi (S. Typhi) on the proliferation of colorectal cancer cells under different culture conditions. Ultracentrifugation was used to extract OMV secreted by S. Typhi in normal, acid, hyperosmotic, oxidative LB medium, and LPM medium. The morphology, particle size and protein distribution of OMV under different conditions were compared. The colorectal cancer cells HT-29, SW480 and CT-26 were cultured in vitro. CCK-8 assay was used to detect the effect of OMV on the proliferation of colorectal cancer cells, and the colony formation experiment was used for further verification. The cell cycle was analyzed by flow cytometry. The toxicity of OMV in mice was evaluated through body weight and hematoxylin-eosin (HE) staining of liver and kidney tissues. The results showed that there was no significant difference in morphology and size of OMV under different culture conditions. Compared with other culture conditions, S. Typhi could secrete more OMV under hyperosmotic stress. In normal and acid LB medium the relative molecular weight of proteins in OMV was 37×10³—72×10³, in hyperosmotic and oxidative LB medium it was 25×10³—72×10³, and in LPM medium it was 8×10³—55×10³. OMV secreted in LPM medium could significantly inhibit the proliferation of SW480 and CT-26 cells, and arrest the cell cycle of SW480 cells in G2/M phase, without obvious hepatorenal toxicity to mice. It is concluded that OMV secreted by S. Typhi in LPM medium exhibit a direct inhibitory effect on the proliferation of colorectal cancer cells (SW480, CT-26), and it is expected to be a therapeutic drug for the treatment of colorectal cancer.