BACKGROUND: Microbial transformation of phytosterols has been applied to produce androst-4-ene-3,17-dione (AD). However, low substrate solubility and product inhibition are major problems to be solved. Biphasic systems are efficient strategies, and ionic liquids (ILs), as one of the phases in biphasic systems, are promising alternatives to traditional organic solvents due to environmental constraints. RESULTS: In this work, nine water-immiscible ILs were used in phytosterols side-chain cleavage by Mycobacterium sp. MB 3683. The solubilities of phytosterols and AD in these ILs and distribution coefficients of AD between water and ILs were first measured. [PrMIM][PF6] was determined to be a suitable candidate to establish a biphasic IL-aqueous system. Under optimal conditions, i.e. inoculum size of 12% with 48 h age, IL addition at 84 h, and phase ratio of 20: 1 (v/v, aqueous/IL), AD production reached 2.23 g L-1 after 5 days of biotransformation with substrate concentration of 5 g L-1 in a scaled-up biphasic system of 200 mL. This bioconversion conducted in a biphasic IL-aqueous system was more efficient than that in a monophasic aqueous system. CONCLUSION: Owing to the environmental advantages of ILs and the above positive results, biphasic IL-aqueous systems have the potential to be applied in phytosterols side-chain cleavage. (C) 2015 Society of Chemical Industry