To optimize a bioreactor callus culture system for Rhodiola sachalinensis, several physicochemical factors affecting callus growth and bioactive compound accumulation were studied using a 3-L, balloon-type airlift bioreactor system. A bioreactor with a sparger size of 3.5-cm diameter was optimal for the callus culture of R. sachalinensis. Suitable light intensities for R. sachalinensis callus growth and biosynthesis of various kinds of bioactive compounds (salidroside, polysaccharides, phenolics, and flavonoids) were different. Maximum productivity of all bioactive compounds was found at a light intensity of 30 μmol m−2 s−1. Callus growth and production of salidroside, polysaccharides, and flavonoids were stimulated with sucrose at a concentration of 30 g L−1, but 50 g L−1 was favorable for the production of phenolics. Salidroside accumulation was promoted when MS medium salt strength increased to 1.5 × MS, 1 × MS was best for the production of polysaccharides and flavonoids, and 0.5 × MS was proper for phenolic production. A kinetics study indicated that a culture duration of 20–25 d was appropriate for the mass production of bioactive compounds from R. sachalinensis callus in a bioreactor system. Callus culture in a bioreactor can be an alternative method for producing materials from R. sachalinensis for commercial drug production.