The use of low-cost substrates represents one key issue to make single cell oil production sustainable. Among low-input crops, Arundo donax L. is a perennial herbaceous rhizomatous grass containing both C5 and C6 carbohydrates. The scope of the present work was to investigate and optimize the production of lipids by the oleaginous yeast Cutaneotrichosporon curvatus from undetoxified lignocellulosic hydrolysates of steam-pretreated A. donax. The growth of C. curvatus was first optimized in synthetic media, similar in terms of sugar concentration to hydrolysates, by applying the response surface methodology (RSM) analysis. Then the bioconversion of undetoxified hydrolysates was investigated. A fed-batch process for the fermentation of A. donax hydrolysates was finally implemented in a 2-L bioreactor. Under optimized conditions, the total lipid content was 64% of the dry cell weight and the lipid yield was 63% of the theoretical. The fatty acid profile of C. curvatus triglycerides contained 27% palmitic acid, 33% oleic acid and 32% linoleic acid. These results proved the potential of lipid production from A. donax, which is particularly important for their consideration as substitutes for vegetable oils in many applications such as biodiesel or bioplastics.