Objective: The incidence of colorectal cancer has increased dramatically in recent years. One of the most important causes of colorectal cancer is infection with a specific type of Escherichia coli bacterium that has polyketide synthase (pks) gene, which produces a secreted toxin named colibactin that changes intestinal microflora condition for its pathogenicity and by interrupting the cell cycle triggers the development of colorectal cancer. Due to the drug resistance of this bacterium, it is necessary to produce new antimicrobial agents such as nanoparticles. Therefore, this study aimed to investigate the effect of titanium dioxide nanoparticles on the expression of clb genes of E. coli responsible for colorectal cancer. Methods: The synthesis of titanium dioxide nanoparticles was done by Bacillus tequilensis that was identified by sequencing 16S rRNA. Titanium dioxide nanoparticles were examined by ultraviolet spectroscopy, X-ray diffraction, infrared spectroscopy, and scanning electron microscopy Real-Time PCR was used to analyze the effect of nanoparticles on clb expression. Human embryonic kidney cells 293 (HEK 293) cell line was treated with different concentrations of titanium dioxide nanoparticles for 72 hours and then cell viability and cytotoxicity were determined MTT method. Results: The synthesized nanoparticles were often spherical between 35.76-78.17 nm with absorption at 350 nm. RT PCR showed a 20-fold reduction in the expression of clbB and clbN genes. MTT showed that cell viability, increased after 72 hours that depends on titanium dioxide concentration. Conclusion: The results obtained in this study indicate that the synthesized nanoparticles could reduce the expression of clbB and clbN genes of E. coli with the lowest cytotoxicity. Therefore, it can be considered for the treatment of colon cancer caused by clb gene-positive E. coli.