The enzymatic hydrolysates of the lipid-extracted microalgal biomass residues (LMBRs) from biodiesel production were evaluated as nutritional sources for the mixotrophic growth of Chlorella vulgaris and lipid production at different temperature levels and substrate concentrations. Both parameters had a significant effect on cell growth and lipid production. It was observed that C. vulgaris could grow mixotrophically in a wide range of temperatures (20∼35 °C). The optimal temperature for cell growth and lipid accumulation of the mixotrophic growth of C. vulgaris was between 25 and 30 °C. The neutral lipids of the culture at 25 °C accounted for as much as 82 % of the total lipid content in the microalga at culture day 8. Fatty acid composition analysis showed that the increase of saturated fatty acids was proportional to the increase in temperature. The maximum biomass concentration of 4.83 g/L and the maximum lipid productivity of 164 mg/L/day were obtained at an initial total sugar concentration of 10 g/L and an initial total concentration of amino acids of 1.0 g/L but decreased at lower and higher substrate concentrations. The present results show that LMBRS could be utilized by the mixotrophic growth of C. vulgaris for microalgal lipid production under the optimum temperature and substrate concentration.