Physiological impact of different exposure periods (1, 2 and 3 h) of UV-C (253.7 nm) on alkaloids and hyoscyamine production in 21-days aged callus of Egyptian henbane was investigated. Results showed that the concentration of total alkaloids was increased by 2.7 and 2.3 times after exposure to UV-C for 2 or 3 h, respectively more than unstressed callus. Also, data of HPTLC revealed that 2 h exposed-callus accumulated the highest amount of hyoscyamine which similar to the amount in wild leaves but it was more 3.1 times than control. Increment of alkaloids concentration was attributed with high accumulation of non-enzymatic antioxidants as free amino acids, free phenolics and glutathione and increased of peroxidase and superoxide dismutase activity as well as reduction of H₂O₂ concentration. Also, 2 h exposed-callus to UV-C showed high expression of electrophoretic protein bands with molecular weights 109, 42 and 29 KDa. All exposure times to UV-C increased both fresh and dry weight of callus compared to unstressed one. Short exposure time of UV-C enhanced the amount of Chl. a and b in callus than other treatments. All exposure periods of UV-C decreased the concentration of carotenoids, anthocyanine, proline and total carbohydrates than control. Long exposure period of UV-C modified the callus cells shape by induction large spherical cells and lysigenous intercellular spaces. It can be concluded that 2 h exposure time of UV-C is effective treatment for enhancement both alkaloids and hyoscyamine content in callus of Egyptian henbane.