Purpose: Radiation-induced bystander effect (RIBE) was considered being related tightly with the radiation-induced secondary-cancer beyond the irradiated area in radiotherapy.Materials and methods: The protective effect of CO (released from carbon monoxide release molecule 2, CORM-2) was assessed in a co-cultured system, which the irradiated cells was trypsinized after irradiation and mixed with the non-irradiated bystander cells for a further culture.The expression of 53 BP1 and micronucleus were employed to test the genetic damage of bystander cells.In the study of mechanism, a method of medium transfer was used for the RIBE signaling assay.Modulation of CO on the signal (nitric oxide, NO) release and action was investigated by the measurement of DNA damage and NO with fluorescent probes.Results: Low concentration (14 μM) CO could protect effectively the bystander from the damage of irradiated cells, and more important that CO treatment did not affect the direct damage effect of irradiation.Mechanism study showed that CO did not inhibit or decreased the signal NO production and release from the irradiated cells, but CO weakened the sensitivity of bystander cells to NO and the production of reactive oxygen species was also down-regulated by CO.Conclusions: Protection of low concentration CO on the bystander cells is very important for the irradiation protection in radiotherapy.The treatment with CO might decrease the ratio of secondary cancer in the survivals after radiotherapy.