Extraction of oil from Portulaca oleracea seeds via supercritical fluid was carried out for the first time. Response surface methodology was applied to evaluate the effect of operating variables, namely pressure, temperature, particle size and extraction time. Optimum conditions were found to be pressure of 23.50 MPa, temperature of 333 K, particle size of 0.8 mm, and dynamic extraction time of 210 min. The highest recovery achieved was 91.88 1.60%, under the optimum conditions, and 92.94%, from RSM. The fatty acid content of the oil was found to be composed of unsaturated (79%) and saturated (20.7%) compounds. Major fatty acids included linolenic, linoleic, and oleic acids. Experiments were performed on physicochemical properties of the oil samples; these included saponification, iodine, peroxide and acid contents, specific extinctions, total phenol content, antioxidant activity, TGA and stability tests. The results indicated high levels of omega-3, making it a good candidate for pharmaceutical and food industries.