Several batch tests and pilot-scale investigations on biological denitrification with isopropanol were performed. Isopropanol was converted to acetone by microbial oxidation during denitrification. Isopropanol itself little contributed to denitrification in practice while the converted acetone played a role of a main hydrogen donor. A larger quantity of nitrite intermediate was formed by using methanol compared to the case of isopropanol. The measured requirement of isopropanol was 2.0 mg mg−1 NO3-N, and was 2/3 of methanol. The oxygen equivalent of isopropanol for nitrate removal was almost the same as that of methanol. The denitrifier net growth yield for isopropanol was greater than for methanol. In order to maximize the denitrification rate, it is essential to convert isopropanol to acetone rapidly by accurate dosing for nitrogen load because the denitrification rate was accelerated by using acetone only. Excessive dose of isopropanol can cause a decrease in the denitrification rate as well as an increase of BOD in the effluent.