Background: Ferula gummosa Boiss. is a medicinal and economical plant belonging to Apiaceae family. It is a native herb of Iran and is in serious danger of extinction. Objective: Gamma irradiation is an environmental stress and can consider as a new tool to change the content of secondary metabolites in medicinal plants. The effect of gamma irradiation on phenolic content and radical scavenging activity of F. gummosa callus was investigated in this research. Methods: Embryogenic axes were removed from seed coats and were cultivated on 1⁄2 Murashige and Skoog medium. After 2 weeks, roots were used as source of plant material for callus induction. Root explants were transferred into MS medium containing NAA (1.5, 3 and 4.5 mg/L) and BA (0, 0.5, 1 and 2 mg/L), and the best medium of callus establishment was selected for gamma irradiation. Sub-cultured calli were irradiated at different doses of gamma irradiation from 0 to 25 Gray (Gy). Results: The obtained data showed that MS medium supplemented with 1.5 mg/L NAA and 2 mg/L BA was the best medium for callus induction. Under gamma irradiation, a significant decline was observed in the fresh and dry weight of irradiated-calli as compared to control one. Phenolic content increased under gamma irradiation especially at 20 and 25 Gy, and the best dose was 20 Gy according to growth response. Aqueous extract showed higher phenolic content than methanol extract. The 50% inhibitory concentration (IC50) obtained in the DPPH radical test decreased significantly under gamma irradiation espacially at 20 and 25 Gy. Conclusion: According to the obtained results, gamma radiation could increase radical scavenging activity by induction of phenolic compounds, and the aqueous extract identified as a proper extraction solvent for free radical scavenging activity in F. gummosa.