Mountain ash (Sorbus commixta Hedl.), is an important medicinal woody plant in Korea used for the treatment of cough, asthma, and other bronchial disorders. Unfortunately, the species is gradually disappearing due to over exploitation of the natural habitats. Tissue culture is a common and powerful technique for the clonal mass propagation which can be adapted to Mountain ash for ecosystem restoration. In an effort to establish a micropropagation method at the stage II for proliferation of shoots, Mountain ash was micropropagated under the so called a photo-autotrophic or photo-mixotrophic culture conditions. Micro-shoots of Mountain ash at the micropropagation stage II were cultured under two levels each of medium sucrose concentration (0 and 30 g l^-1), photosynthetic photon flux density (PPFD, 50 and 200 µmol m^-2 s^-1), and CO₂ concentration (350 and 1,000 µmol mol^-1). Axillary shoots originated from in vitro-grown plantlets, were stuck into and cultured on 50 ml per container of agar-solidified half-strength Murashige and Skoog (MS) medium with 2.0 mg l^-1 indole-3-butyric acid (IBA). Tissue water content, leaf length, and total chlorophyll content increased in plantlets cultured in the medium without sucrose (a photo-autotrophic condition). High PPFD (200 µmol m^-2 s^-1) promoted axillary shoot length, number of nodes, axillary shoot fresh weight, axillary shoot dry weight, leaf length, leaf width, and total chlorophyll content especially in the photo-autotrophic condition. High CO₂ concentration (1,000 µmol mol^-1) increased axillary shoot length, number of axillary shoots, number of nodes, stem diameter, axillary shoot fresh weight, and axillary shoot dry weight. The treatment with a high PPFD (200 µmol m^-2 s^-1), a high CO₂ concentration (1,000 µmol mol^-1), and without supplementation of sucrose to the medium (a photo-autotrophic condition) was better for growth and development in terms of number of nodes, tissue water content, leaf length, leaf width, and total chlorophyll content than those in the other treatments.