BACKGROUND: 10-Hydroxy-cis-12-octadecenoic acid (10-HOE, 10-OH C18:1), an emerging functional fatty acid, has anti-fungal and anti-inflammatory effects. 10-HOE is synthesized by bacterial 10-linoleic acid hydratase (10-LHT) with linoleic acid as the substate. However, the characterization of 10-LHT and its targeted synthesis of 10-HOE have been rarely reported. In this study, the recombinant 10-LHT from Lactiplantibacillus plantarum ZS2058 was characterized, and the biocatalysis of 10-HOE using crude enzyme was optimized.RESULTS: The recombinant 10-LHT catalyzed the conversion of linoleic acid (C18:2) to 10-HOE as identified using gas chromatography-mass spectrometry (GC-MS). It showed a molecular weight of about 70 kDa on sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), and was a flavin adenine dinucleotide (FAD)-dependent enzyme. The activity of 10-LHT was optimal at pH 6.5 and 25 °C, and it was pH-stable but thermo-sensitive. The optimal condition for the 10-HOE biosynthesis using crude enzyme was 5 g L −1 linoleic acid (C18:2), 148.0 U mL −1 10-LHT, 0.05 mmol L −1 FAD, 2% methanol and 100 mmol L −1 sodium chloride at 25 °C and pH 6.5. A conversion yield of 47.8 ± 1.5% and the corresponding 10-HOE concentration of 2.4 ± 0.1 g L −1 were achieved at 48 h under the optimal reaction conditions. CONCLUSION: This work achieved the highest conversion yield of 10-HOE with the highest substrate concentration, and provides some useful information for the industrial production of 10-HOE.