Lipase of Acinetobacter radioresistens was produced using Tween 80 as the carbon source. Compared with olive oil, the hydrolysis rate of Tween 80 in the fermentation broth was much slower. Taking advantage of the slow hydrolysis rate, Tween 80 could provide oleic acid for cell growth and lipase production through a mode of controlled release, and the known repression of lipase synthesis by oleic acid could be avoided. The optimal lipase yield obtained with batch culture in a 2.5-L tank fermentor was 25 U/mL with a fermentation time of 6 h, when using 0.3% (v/v) Tween 80. The existence of an optimal yield with respect to concentration of Tween 80 was a result of the compromise between cell growth and the repression of lipase synthesis. To further the lipase productivity, a fed-batch culture with the pH-stat feeding method was employed, and the lipase productivity achieved was 120 U/mL in 16 h. Various strategies for production of A. radioresistens lipase were compared.