Lipase usually has little interesterification activity in organic solvents, probably owing to the absence of an oilwater interface. Lipases were processed in a two-phase hydrocarbon-water system that had an oil-water interface. Crude lipase (from Rhizopus japonicus) in a buffer and a small volume of aliphatic hydrocarbon as an oil phase were mixed and then lyophilized to remove the aqueous and oil phases. The interfacially processed lipase has a remarkable interesterification activity in n-hexane compared to crude native lipases. We postulate that this activation is caused by the oil-water interface, i.e., the interface between hydrocarbon and water makes the lipase lid open and enables the lipase to work effectively in n-hexane. Several different hydrocarbons were investigated as an oil phase, and n-tetradecane was found to be the best for interesterification. Activated lipase was successfully inactivated in a water suspension without an oil-water interface, and the inactivated lipase could be reactivated. We demonstrated that the oil (hydrocarbon)-water interface induced reversible activation to lipase for interesterificaiton.