The phospholipid fatty acids composition of gum resin of Styrax benzoin was successively macerated with chloroform over 2 days. The exhaustively extracted at room temperature with CHCl3 was evaporated to obtain the crude extract. Vacuum liquid column chromatography, thin layer chromatography and HPLC were performed to obtain two pure compounds. Both of compounds were elucidated and identified using the spectroscopic methods. We employed lipopolysaccharide (LPS)-induced nitric oxide inflammatory model in RAW264.7 macrophage cells. Successfully repeated chromatographic techniques with chemical methods resulted in purification and structure evaluation of two new C18 unsaturated hydroxy fatty acids from the gum resin of Styrax benzoin. Their structures were determined to be (3R,4E,6E)-3-Hydroxy-4,6-octadecadienoic acid (1) and (3R,9E,11E,15E)-13-Hydroxy-9,11,15-octadecatrienoic acid (2) according to different spectroscopic data. The configuration of ethylenic moiety were determined to be an E- stererostructures by comparison of coupling patterns of related proton signals in the 1H-NMR and NOESY experiments. Other supporting results for the stereogenic centers for C-3 of compound (1) and C-13 of (2) by helping of Newman projection. Finally, the absolute stereochemical configuration was determined by using the modified Mosher method. Compound 2 showed a strong anti-inflammatory effect, as revealed by its concentration-dependent inhibition of LPS-induced NO release with IC50 of 13.5 µM.