Abstract: Mucor circinelloides has the ability to use a wide range of carbon sources to accumulate lipids. The different carbon sources have significantly influenced the total lipids through different metabolic pathways in oleaginous organisms. In the present study, the effects of exogenous oil (soybean oil) at different concentrations as an alternative carbon source on its fungal growth and lipid accumulation were investigated without changing fermentation culture conditions and the original C/N ratio, while using 100% glucose as a control. Total lipids and their fatty acid composition were determined by gas chromatography (GC). Moreover, the expression levels of desaturases genes involved in fatty acid biosynthesis were investigated in the studied strains using real-time fluorescence quantitative PCR. The results showed that a concentration of 35 g/L of glucose mixed with 24.3 g/L of soybean oil were the optimal carbon sources with constant carbon to nitrogen ratio. The biomass, lipid content and lipid yield of M. circinelloides grown in this medium increased rapidly in the early stage of fermentation, and the growth trend gradually slowed down in the late stage of fermentation, decreasing slightly after 72 h of fermentation. It was found that the highest biomass, total lipids, and lipid yields were significantly increased at 19.60 g/L, 42.62%, and 8.35 g/L (33.15%, 23.8%, and 65.10%), respectively. In contrast, the control group (those cultured on glucose as a single carbon source) was only 14.72 g/L, 34.41%, and 5.06 g/L, respectively. The critical step was in which α-linolenic acid (ALA) was stored from the exogenous oil in fungal cells supplying enough soybean oil with mixed substrates for fatty acids synthesis. The overexpression of the △12-desaturase gene in WJ11 significantly (P<0.05) increased, while the overexpression of △9- and △6-desaturases genes was significantly (P<0.05) decreased. This study could provide important insights for further investigation of the effect of other exogenous oils on lipid production by M. circinelloide and other oleaginous fungi.