Omega-3 fatty acids, including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have beneficial effects on human health. The probiotic bacterium Escherichia coli Nissle is unable to produce either EPA or DHA. Escherichia coli Nissle was transformed with the pfBS-PS plasmid carrying the EPA/DHA gene cluster, previously isolated from a marine bacterium. The transgenic E. coli Nissle produced EPA when grown at 10ºC (16.52 ± 1.4 mg g(-1) cell dry weight), 15ºC (31.36 ± 0.25 mg g(-1) cell dry weight), 20ºC (13.71 ± 2.8 mg g(-1) cell dry weight), 25ºC (11.33 ± 0.44 mg g(-1) cell dry weight) or 30ºC (0.668 ± 0.073 mg g(-1) cell dry weight). Although DHA was also produced at all these temperatures, it comprised less than 0.2% of total extracted fatty acids. Transcriptomic analysis using Reverse Transcription qPCR showed upregulation of the entire gene cluster in E. coli Nissle. Among EPA/DHA genes, pfaB, pfaC and pfaD were overexpressed (expression ratio of 181.9, 39.86 and 131.61, respectively) as compared to pfaA (expression ratio of 3.40) and pfaE (expression ratio of 4.05). The EPA/DHA-producing probiotic E. coli Nissle may be used as a safe, alternative and economic source for the industrial and pharmaceutical production of EPA and DHA.