The in vitro anti-inflammatory activity profile of the Nerium oleander flower EtOH extract/its subextracts (n-hexane, CH2Cl2, EtOAc, remaining H2O) were evaluated on LPS induced Raw 264.7 macrophages. The effects of the crude EtOH extract and its subextracts on nitric oxide (NO) production and cell viability were determined. The most active subextract was determined to be the EtOAc subextract without exerting any toxicity towards Raw 264.7 macrophages. This subextract significantly inhibited NO production of Raw 264.7 macrophages after LPS induction (62.56 +/- 1.91% at 200 mu g/mL concentration). The levels of iNOS were reduced up to 67.50%. Moreover, this subextract slightly reduced the phosphorylation levels of MAP kinases (p-ERK, p-JNK, p-38). The highest inhibition was observed for ERK phosphorylation, which was inhibited by 20.53% at 200 mu g/mL concentration. Through activity-guided fractionation procedures, kaempferol, kaempferol 3-O-beta-glucopyranoside and chlorogenic acid were isolated as the main active components. The structures of the active compounds were determined by 2D-NMR techniques and HRMS analysis. All compounds significantly inhibited NO productions. Results of the present study supported the traditional use of N. oleander flowers to treat inflammatory complaints.