An efficient protocol for in vitro propagation of Digitalis trojana Ivan. was developed via adventitious shoot regeneration. Leaf explants were cultured on MS which were supplemented with different concentrations of NAA (0.1, 0.5, 1.0 mg/ml) and BAP (0.1, 0.5, 1.0, 3.0, 5.0 mg/ml) for shoot formation. Adventitious shoots were formed on leaf explants within three weeks in culture. The best shoot proliferation was observed among explants cultured on MS medium with 0.1 mg/ml NAA + 3.0 mg/ml BAP. Regenerated shoots were multiplicated by subculture. Then they were cultured on MS with 0.1% (w/v) activated charcoal for root formation. All of the in vitro regenerated plantlets were successfully acclimatized ex vitro and then grown healthy.