As summarized in Table 2, the fermentation of glucose (positive control) showed the good cell growth of 7.7 g/L, high fatty Trametinib production (the content, production and yield of 40.3 wt%, 3.0 g/L and 0.18 g/g, respectively) and high neutral lipid production (the content and yield of 64.7 wt%, 0.34 g/g, respectively). The batch cultures using CBHs, with or without acid pretreatment, produced similar fatty acids (the content and productivity of about 35 wt% and 0.022 g/L/h, respectively), neutral lipids (the content and productivity of 46–48 wt% and 0.022 g/L/h, respectively), and the ratios of total fatty acid to neutral lipid (70–75%). The great accumulation of fatty acids and neutral lipids using CBHs was achieved despite the fact that the lipid accumulation from CBHs was lower than that from glucose. In addition, it was shown from the experiments that the hydrolysis of CB without acid pretreatment was preferable considering the treatment costs. Taken together, the CBH with direct cellulase hydrolysis was a favorable carbon source for heterotrophic C. protothecoides to produce lipids with higher yield and productivity. The glucose depletion in batch culture indicated that the supplement of higher concentration of sugars in the basal medium or nutrient feeding could further improve the lipid accumulation of C. protothecoides.