Epoxy fatty acids (EFAs), which exist in the human body, are signaling molecules that maintain homeostasis. They are involved in anti-inflammation and are precursors of dihydroxy fatty acids. EFAs derived from the C20 polyunsaturated fatty acid (PUFA) arachidonic acid by lipoxygenases, such as 5,6-epoxy-7E,9E,11Z,14Z-eicosatetraenoic acid and 8,9-epoxy-5Z,10E,12E,14Z-eicosatetraenoic acid, are found in humans and mice, respectively. However, EFAs derived from C18 PUFAs by lipoxygenases have not been identified to date. In this study, the putative lipoxygenase gene of Spingopyxis macrogoltabida was cloned and expressed in Escherichia coli. The activity and catalytic efficiency (kcat/Km) of the recombinant enzyme were the highest for linoleic acid among the C18 PUFAs, including also α-linolenic acid and γ-linolenic acid. The product obtained from the conversion of linoleic acid by the putative lipoxygenase was identified as 9-hydroxy-10E,12Z-octadecadienoic acid (9-HODE) by high-performance liquid chromatography using 9-HODE and 13-hydroxy-9Z,11E-octadecadienoic acid (13-HODE) standards. These results indicate that the enzyme is a linoleate 9-lipoxygenase. The enzyme converted linoleic acid, α-linolenic acid, and γ-linolenic acid into 9-HODE, 13-hydroxy-9Z,11E,15Z-octadecatrienoic acid, and 9-hydroxy-6Z,10E,12Z-octadecatrienoic acid, respectively. Moreover, the enzyme also converted the three C18 PUFAs into 9,10-epoxy-11E,13E-octadecadienoic acid, 12,13-epoxy-8E,10E,15Z-octadecatrienoic acid, and 9,10-epoxy-6Z,11E,13E-octadecatrienoic acid, respectively, which were identified as new EFAs by liquid chromatography-mass spectrometry/mass spectrometry. To our knowledge, this is the first report on the biosynthesis of EFAs from C18 PUFAs via a lipoxygenase.