The study examined tyrosol glucosyltransferase activity and the efficiency of salidroside production in natural and transformed root cultures of Rhodiola kirilowii (Regel) Regel et Maximowicz. Neither enzyme activity nor salidroside accumulation were detected in natural and transformed root cultures maintained in media without tyrosol. To induce TGase activity in biotransformation reactions, tyrosol was added to natural and transformed root cultures on the day of inoculation. The first peak of TGase activity (0.23 U/μg) was detected on day 9 in natural root culture, accompanied by the highest salidroside content (15.79 mg/g d.w.), but TGase activity was highest (0.27 U/μg) on day 15. In transformed root culture, day 18 showed the highest TGase activity (0.15 U/μg), which coincided with the highest salidroside content (2.4 mg/g d.w.). Based on these results, tyrosol was added to the medium on the days of highest previously detected activity of TGase: day 15 for natural root cultures and day 18 for transformed root cultures. This strategy gave significantly higher yields of salidroside than in the cultures supplemented with tyrosol on the day of inoculation. In natural root culture, salidroside production reached 21.89 mg/g d.w., while precursor feeding in transformed root cultures caused a significant increase in salidroside accumulation to 7.55 mg/g d.w. In all treatments, salidroside production was lower in transformed than in natural root cultures.