Ricinoleic acid (RA), a hydroxyl fatty acid, is suitable for medical and industrial uses and is produced in high-oil-accumulating organisms such as castor bean and the ergot fungus Claviceps. We report here the efficient production of RA in a transgenic diatom Chaetoceros gracilis expressing the fatty acid hydroxylase gene (CpFAH) from Claviceps purpurea. In transgenic C. gracilis, RA content increased at low temperatures, reaching 2.2 pg/cell when cultured for 7 d at 15 °C, without affecting cell growth, and was enhanced (3.3 pg/cell) by the co-expression of a palmitic acid-specific elongase gene. Most of the accumulated RA was linked with monoestolide triacylglycerol (ME TAG), in which one RA molecule was esterified to the α position of the glycerol backbone and was further esterified at its hydroxy group with a fatty acid or second RA moiety, or 1-OH TAG, in which RA was esterified to the glycerol backbone. Overall, 80% of RA was accumulated as ME TAGs. Furthermore, exogenous RA-methyl ester suppressed the growth of wild-type diatoms in a dose-dependent manner and was rapidly converted to ME TAG. These results suggest that C. gracilis masks the hydroxyl group and accumulates RA as the less-toxic ME TAG.