Bacterial strain JYR-1, which utilizes high concentrations (up to 100 mM) of trans-anethole as the sole source of carbon and energy, was isolated from soil. It grew to OD(600)(nm) = 2.6 with a doubling time of 8 h when grown on 20 mM trans-anethole. Strain JYR-1 was identified as Pseudomonas putida based on the partial gene sequence of its 16S rDNA. Elution profiles of culture extracts were examined by high-performance liquid chromatography and showed that four metabolites were produced from the bacterial culture containing trans-anethole that were not detected in control experiments. LC-MS analysis showed molecular weights of 138.2, 164.5, 164.3, and 152.3. The metabolites with molecular weights at 152.3 and 138.2 were confirmed to be p-anisic acid and p-hydroxybenzoic acid, respectively, when compared with HPLC retention times and molecular weights of authentic compounds. The metabolites with molecular weights at 164.5 and 164.3 were further analyzed by NMR and were proved to be stereoisomer syn- and anti-anethole epoxides. Therefore, strain JYR-1 most likely initiates the metabolism of trans-anethole through the formation of epoxides on the propene group of the compound.