Abstract The total soluble sugar content and antioxidant enzyme activities were studied for the first time during axillary shoot formation in Magnolia × ‘Spectrum’ in vitro in response to BAP (0.3 mg l−1), different levels of gibberellic acid (GA3; 0.0, 0.1, 0.5, 1.0 mg l−1), sucrose (20 and 30 g l−1) and nitrogen salts (KNO3/NH4NO3; 100/100% and 75/50% relative to MS medium). Among various GA3 and sucrose/nitrogen salts ratios, the most effective axillary multiplication (5.9 shoots/explant) and leaf formation (25.7 leaves per multiplied clumps) were obtained after addition of GA3 at 0.1 mg l−1 to a BAP medium containing 20 g l−1 sucrose and reduced levels of nitrogen salts (75% KNO3 and 50% NH4NO3). The addition of GA3 to the BAP medium enhanced shoot formation by 36% and leaf formation by 27%. The highest shoot formation capacity of M. × ‘Spectrum’ in vitro coincided with enhanced levels of soluble sugar and peroxidase (POD) activity. Increasing GA3 concentration from 0.1 to 1.0 mg l−1 in the above medium resulted in inhibition of shoot and leaf formation and a decrease in the soluble sugar content. The influence of GA3 on the activities of catalase (CAT) and POD depended on its concentration and the levels of sucrose and nitrogen salts in the medium. The highest increase in CAT and POD activities, that coincided with the enhanced shoot formation capacity of M. × ‘Spectrum’ in vitro, was observed after addition of GA3 to the medium containing high levels of sucrose and nitrogen salts.