In this paper, a novel strategy for lipid production through coupling oleaginous yeasts and activated sludge biological methods by cultivation of Rhodotorula glutinis in cellulosic ethanol wastewater was studied. Under optimal conditions in wastewater medium (dilution ratio of 1: 2 and glucose supplement of 40 g/L), the maximum biomass and lipid content as well as the lipid yield reached 11.31 g/L, 18.35% and 2.08 g/L, with the associated removal rates of COD, TOC, NH4+-N, TN and TP reaching 83.15%, 81.81%, 85.49%, 70.52% and 67.46%, respectively. Cellulosic ethanol wastewater treated by the anaerobic-aerobic biological process resulted in removal of COD, NH4+-N, TP and TN reaching 67.55%, 94.17%, 90.16% and 48.89%, respectively. The reused water was used to dilute medium of R. glutinis for microbial lipid production reaching 2.38 g/L and caused positive effects on the accumulation of biomass and lipid.