Lipids produced by oleaginous microorganisms are a potential feedstock for biodiesel production and chemical synthesis. Yet, the costs of microbial lipids remain high, partially because the lipid recovery process is tedious and costly. In the present study, enzyme-assisted extraction of lipids from the culture of the yeast Rhodosporidium toruloides was carried out. With a heat pre-treatment with microwave, enzymatic treatment with the recombinant β-1,3-glucomannanase, plMAN5C, and extraction with ethyl acetate, 96.6% of the total lipids were extracted from R. toruloides cells at room temperature and atmospheric pressure directly from the culture without dewatering. Therefore, this process could significantly reduce energy consumption and costs for lipids extraction from the yeast.