In the present work, the inhibitory effect of carbon monoxide (CO), generated by tricarbonyldichlororuthenium (II) dimer [CO-releasing molecule (CORM-2)], on the toxicity of radiation-induced bystander effect (RIBE) after -particle irradiation was studied in a mixed coculture system. CO (CORM-2) treatment showed a significant inhibitory effect to the formation of p53 binding protein 1 (BP1) and micronuclei (MN) induced by RIBE in a concentration-dependent manner, but in the directly irradiated cell population no distinct decreases of BP1 and MN formation were observed. In this mixed coculture system, nitric oxide (NO) or superoxide anion $${(\hbox{ O }}_{2}^{\cdot -})$$ was also proved to mediate the transduction of RIBE by using a NO synthase inhibitor or NADPH-oxidase-specific inhibitor treatment. The elevated $${\hbox{ O }}_{2}^{\cdot -}$$ was attenuated by CO (CORM-2) treatment in the bystander cells as measured by hydroethidine staining and fluorescence assessment. The exogenous NO (sper) or $${\hbox{ O }}_{2}^{\cdot -}$$ (H2O2) was used to mimic NO/O2-mediated RIBE, and CO (CORM-2) treatment also showed a protective effect to cells against the toxicity of these exogenous factors. Considering the inhibitory effect of CO on RIBE and the wide use of CO in therapy of diseases, it is hoped that a low concentration of CO can protect normal tissues against RIBE during radiotherapy.