Abstract: The aim of this study was to identify and analyze the interaction of andrographolide with COX-2, followed by in vitro study of the effect of this compound on COX-2 expression in human fibroblast cells. The molecular modeling study was performed by docking andrographolide to COX-2 enzyme at the site where SC-558, a selective inhibitor of this enzyme, was co-crystallized and compared its interaction to the enzyme with SC-558’s. The inhibition of COX-2 expression was determined by measuring PGE2 production in human fibroblast cells stimulated with LPS with and without andrographolide preincubations. Andrographolide interacted with Arg513 and His90 in the cyclooxygenase site of COX-2 and inhibited PGE2 production in human fibroblast cells (IC50 = 4 μM). These data confirm that andrographolide’s anti-inflammatory activity occurs via inhibition of COX-2 expression.