Echium amoenum Fisch. C.A. Mey. (Boraginaceae) that grows in the northern mountains of Iran is the source of very valuable essential fatty acids such as gamma linolenic acid (GLA) and stearidonic acid (SDA) that are mainly used in the pharmaceutical, medicinal and dietary applications. GLA and SDA are polyunsaturated fatty acids which are used to destroy tumor cells including breast, prostate and pancreatic cancers without harming normal cells. The common technique to extract oil from seeds is the conventional method such as Soxhlet with organic solvents such as n-hexane and petroleum ether. But the pharmaceutical and medicinal applications led to the need of developing better methods of extraction and purification without utilization of toxic organic solvents. Therefore in this study, essential fatty acids containing linoleic, alpha linolenic, gamma linolenic and stearidonic acid were extracted from Echium amoenum (Boraginaceae) seed oil via supercritical carbon dioxide and the results were compared with conventional Soxhlet method. The Response Surface Methodology (RSM) was used to optimize the effective extraction parameters. The chemical composition of recovered oil was analyzed by polar and non-polar GC columns. The experimental results of this study indicated that echium seed contained 25 wt% oil and 92% as the maximum extraction recovery of oil was achieved at optimal operating conditions (43 °C, 280 bar, 1.5 ml/min, 25 min static time and 130 min dynamic time) using pure CO 2 . Applying 2.5 and 5 mole% ethanol as a modifier at similar operating conditions enhanced the recovery to 96 and 112 %, respectively. The statistical analysis demonstrated that all linear terms except static time, quadratic of temperature, pressure and dynamic time as well as the interactions between pressure and temperature, and static and dynamic times have significant effects on oil yield. The results indicated that supercritical fluid extraction is a viable technique for separation of constituents such as palmitic acid (6-7%), stearic acid (3-4%), oleic acid (12-13%), linoleic acid (19-20%), alpha linolenic acid (40-41%), gamma linolenic acid (7-8%) and stearidonic acid (8-9%). Keywords: Echium amoenum ; Gamma linolenic acid; Supercritical extraction; breast cancer; Essential fatty acid , Response Surface design