In this work, methanol in crude glycerol solution was used to assist the lipid production with oleaginous yeast Trichosporon oleaginosus cultivated under non-sterilized conditions. The investigated methanol concentration was 0%, 1.4%, 2.2%, 3.3% and 4.4% (w/v). The results showed that methanol played a significant role in the non-sterilized fermentation for lipid production. The optimal methanol concentration was around 1.4% (w/v) in which the growth of T. oleaginosus was promoted and overcame that of the contaminants. The non-sterilized fed-batch fermentation with initial methanol concentration of 1.4% (w/v) was then performed and high biomass production (43.39 g/L) and lipid production (20.42 g/L) were achieved.