Effects of sugars, mineral salts and plant growth regulators on the development of Stevia shoots cultivated in the roller bioreactor and their production of steviol glycosides (SGs) were investigated. In the medium with fructose or glucose, extension of the shoots and development of their root system were much better than in the medium supplemented with sucrose. Under these conditions, however, accumulation of leaves dry mass decreased, and the content of the SGs in leaves declined. At elevated sucrose concentrations (from 1 to 5%), enhanced development of the root system and an increase in plant dry mass and number of leaf pairs was observed. At the same time, 3% sucrose gave optimal SG accumulation. Twofold elevating the concentration of mineral salts considerably stimulated growth of the shoots, whereas the content of the SGs in their leaves decreased by about order of magnitude. Addition of 0.1 mg/l BA or 6-benzylaminopurine (BA) together with α-naphthaleneacetic acid resulted in an 1.5-fold increase in the number of shoots. However, the shoots grown on the BA-supplied medium displayed a strong inhibition of the development of their root system. When the medium was supplied with gibberellic acid, lengthening of shoots and roots of Stevia were observed. All the plant growth regulators used strongly inhibited production of SGs. The changes in nutrition medium composition had practically no effect on the ratio of individual glycosides in Stevia leaves.