We investigated the response of Mucor rouxii to ethanolic stress conditions. A differential response was found that was dependent on growth phase and ethanol concentration. 5% (v/v) ethanol showed an inhibitory effect on the mycelial growth of all stages. However, the ethanol sensitivity was specifically observed in active growing phases (12 and 21 h-grown cultures), in which the biomass and ratio of unsaturated/saturated fatty acids (UFA/SFA) decreased greatly after ethanol exposure compared to non-ethanol adding culture. With respect to different ethanol concentrations, M. rouxii was tolerant to low ethanol concentrations (about 1–3%, v/v) such that there was not much change in biomass and UFA/SFA ratio, in contrast to the 5% ethanol-added culture. We also showed the molecular basis of this response mechanism, demonstrating that expression of Δ9-, Δ12- and Δ6-desaturase genes, responsible for fatty acid desaturation in M. rouxii, were coordinately down-regulated upon exposure to ethanol stress.