Premna integrifolia Linn. is a medicinal plant used in “Dhasamula” drug preparation of Ayurvedic systems of medicine in the treatment of various ailments like bronchitis, dyspepsia, liver disorders, piles, constipation, hyperlipidemia and fever. The anti-atherosclerotic activity of hydroalcoholic extract (HAE) of root bark of P. integrifolia was evaluated in high fat diet induced atherosclerosis rats. Sixty Wistar rats were divided into six groups: the first group served as control, the second group was fed with high fat diet and the other three groups were fed with high fat diet along with various concentrations of HAE and the last group was treated with atorvastatin for 30 days. Lipid and lipoprotein profile, atherogenic index, and cardiac markers and histopathological evaluation of aorta were determined in high fat diet induced atherosclerosis rats. HAE of P. integrifolia produced a significant and dose-dependent anti-atherosclerotic activity in terms of reduction in lipids and lipoprotein profile, atherogenic index, HMG-CoA reductase activity, marker enzymes such as lactate dehydrogenase (LDH), creatine phosphokinase (CPK), aspartate transaminase (AST), alanine transaminase (ALT) and alkaline phosphatase (ALP), alteration in collagen and calcium contents, mild mineralization and focal rupture of intima and media of aorta was noticed in treated groups as compared to the control. The results suggested that anti-atherosclerotic activity of HAE of P. integrifolia Linn. was due to its modulatory activity on metabolic pathway of lipid. The results contribute to the validation of the traditional use of Agnimantha in high fat diet induced atherosclerosis rats.