The aim of this study was to provide a theoretical basis for a high-energy carbon ion irradiation process for inducing mutations for selective breeding in Isatis indigotica Fort. The experiments were designed to evaluate the effects of different doses of high-energy 12C6+ ions (10-140 Gy) on physiological changes in I. indigotica seedlings. Dry seeds of I. indigotica were irradiated using different doses of 270 MeV energy 12C6+ ion beam, and the response of the subsequent seedlings was monitored using well-established indexes of physiological characteristics. Results showed that optimum results were achieved with a high-dose 12C6+ ion beam at 35 Gy where the activities of superoxide dismutase (SOD) and catalase (CAT) were enhanced. However, excessive irradiation reduced the activities of SOD and CAT. As the dose increased, the activity of peroxidase increased initially and then decreased compared with controls that were not irradiated (0 Gy). The content of malondialdehyde first decreased and then increased with the treatment. Total soluble protein content initially increased and then decreased with increasing doses of radiation; the proline content improved sharply compared to the control. The results of this study suggest that that a low dose of 12C6+ ion beam could enhance the activities of protective enzymes and the levels of proline and soluble protein, and that a dose range of 35–60 Gy is likely to be optimum for inducing useful mutations in I. indigotica for a stable selective breeding program.