Dehydrogenated derivatives of corticosteroids are usually more effective than their precursors in treating diseases. In this study, the toxicity of seven water-miscible ionic liquid and three organic solvents to the biocatalyst Arthrobacter simplex UR016 was tested to evaluate the possibility of biodehydrogenation from 17α-hydroxy-16β-methyl-pregna-4,9(11)-diene-3,20-dione (HMPDD) to 17α-hydroxy-16β-methyl-pregna-1,4,9(11)-triene-3,20-dione (HMPTD) in an ionic liquid-containing system. Although most tested room temperature ionic liquids (RTILs) showed higher toxicities to A. simplex UR016 than organic solvents, bacterial growth was promoted in the presence of [EMIM](L)-Lac or [BMIM](L)-Lac at concentrations below 2.5 mmol/l, especially [EMIM](L)-Lac, presented the lowest toxicity to A. simplex. Following immobilization investigations, a conversion ratio of 89.9 % was achieved with a cell biomass of 10 g/l (dry cell weight/reaction mixture volume) in the polyethylene glycol (PEG)-modified calcium alginate gel bead, a suitable matrix for cell immobilization. Further studies indicated that the conversion ratio can be improved by increasing cell loading to 60 beads per flask (containing 30 ml reaction mixture). Under optimal conditions with a [EMIM](L)-Lac content of 0.3 %, the conversion ratio reached 93.4 %, the highest value ever reported.