Background: Cnidium officinale Makino have been used in traditional medicine in Northeast Asia. Although gamma-ray mutagenesis has been used to develop breeding resources with novel characteristics, research on the radiation sensitivity of C. officinale Makino is limited. Hence, the optimal gamma-ray dosage for mutation breeding in C. officinale Makino was investigated. Methods and Results: Seedstocks were exposed to doses of gamma rays (5 Gy - 50 Gy), and subsequently planted in a greenhouse. After 30 days of sowing, the survival rates and growth decreased rapidly at doses above 20 Gy, while all individuals died at 50 Gy. The median lethal dose (LD50) was 25.65 Gy, and the median reduction doses (RD50) for plant height, number of stems, and fresh weight were 12.81, 9.32, and 23.26 Gy, respectively. Post-irradiation levels of malondialdehyde (MDA), peroxidase (POD), and chlorophyll in the aerial parts of the plant were quantified using spectrophotometry. Relative to the controls, the levels of MDA and POD increased, while the level of chlorophyll decreased at doses ≥ 10 Gy, indicating cellular damage. Conclusions: A dose of 20 Gy was found to be optimal for mutation breeding in C. officinale Makino.