In recent years, VFAs as a novel, inexpensive substrate for biodiesel production have become a hot research topic. The effect of pH on lipid production by Rhodotorula glutinis when using acetate as a substrate was studied. The optimized initial acetate concentration and C/N ratio were 23 g/L and 350, respectively. In 1‐L bioreactors containing an initial acetate concentration of 23 g/L, the most suitable pH for cell growth was 6.6, while pH 7.0 was found optimal for lipid production. In the pH‐stat fed‐batch fermentation, an increased biomass yield of 67.2 g/L was obtained at pH 6.6, while an increased lipid titer of 29.3 g/L was obtained at pH 7.0. When using a two‐stage pH regulation culture strategy, the biomass yield, lipid titer, and lipid content reached 68.2 g/L, 35.8 g/L, and 52.5%, respectively. Both the biomass yield and lipid production were obviously enhanced. Compared with the batch culture, the biomass yield, lipid titer, and lipid content were increased 15.6‐fold, 19.1‐fold, and 21.0%, respectively, by using the two‐stage pH regulation culture strategy. The composition of the main fatty acids was compared when acetate or glucose was used as the substrate. The composition of the lipids produced using both substrates was similar to that of palm oil, indicating that lipids produced by R. glutinis in the presence of acetate showed promise for use as a biodiesel feedstock.