Mucor rouxii CFR-G15, a locally isolated phycomycetous fungus, on cultivation at room temperature produced more than 30% (w/w) lipid in their dry cell weight, in which 14.2% accounted to be GLA content of the total fatty acids. It was observed that when incubation temperature lowered at 14°C, GLA content of the mycelium increased significantly (P<0.05) from 14.2% to 21.97%. In order to optimize the cultural conditions for high biomass and lipid production with high GLA content, the fungus was grown in association of two different temperatures and supply of additional glucose in culture medium. Maximum lipid and GLA were obtained 23.56 and 19.5% respectively, when the culture was grown at 28°C for four days and followed by addition of glucose (5%), and lowered the incubation temperature to 14°C for another four days. The presence of GLA in the oil obtained from M. rouxii CFR-G15 was confirmed by the gas chromatography-mass spectrometry. Gamma linolenic acid (GLA, n-6) is gaining importance in pharmaceutical and nutraceutical industries because of clinical evidence demonstrated that it has various beneficial effects in human health. In this paper temperature played a major role in enhancing the GLA content which has been described.