To provide some pharmacological evidence for its clinical use in inflammatory diseases, anti-inflammatory effect of the aqueous extract from Radix Ophiopogon japonicus (ROJ-ext), a traditional Chinese herb, was examined in mouse and rat models. ROJ-ext significantly inhibited xylene-induced ear swelling and carrageenan-induced paw edema in mice when given orally at doses of 25 and 50 mg/kg. Moreover, ROJ-ext also remarkably suppressed carrageenan-induced pleural leukocyte migration in rats and zymosan A-evoked peritoneal total leukocyte and neutrophil migration in mice, while had no obvious effect on pleural prostaglandin E2 level. Furthermore, two active compounds were isolated from ROJ-ext and identified as ruscogenin and ophiopogonin D. As the results, ROJ-ext, ruscogenin and ophiopogonin D dose-dependently reduced phorbol-12-myristate-13-acetate (PMA)-induced adhesion of HL-60 cells to ECV304 cells, with IC50 of 42.85 microg/ml, 7.76 nmol/l and 1.38 nmol/l, respectively. However, they showed no inhibitory effect on PMA-induced cyclooxygense-2 (COX-2) mRNA expression in ECV304 cells. Ruscogenin and ophiopogonin D also notably decreased zymosan A-induced peritoneal leukocyte migration, in comparison with ROJ-ext. These results demonstrate that ROJ-ext presents remarkable anti-inflammatory activity and ruscogenin and ophiopogonin D are two of its active components, which supported its traditional use in the treatment of various diseases associated with inflammation.