Atherosclerosis (AS) is a common pathogenesis of cardiovascular diseases. Qingre Huoxue Decoction (QRHX) is an herbal formula used for the prevention and treatment of AS. However, the potential mechanism of QRHX is not clear.In our study, RNA sequencing combined with preclinical models were used to analyse the effect and mechanism of QRHX for the treatment of AS.For in vivo studies, ApoE-/- mice were fed with a high-fat diet to induce AS. We measured weight, blood lipid, inflammatory cytokines, lipid deposition, plaque, and the M1/M2 macrophage. For in vitro studies, RAW264.7 were induced by lipopolysaccharides and treated with different concentrations of QRHX. We focusd on the relationship between QRHX, the NF-κB pathway, and macrophage polarisation, and performed simultaneous RNA sequencing both in vivo and in vitro.In vivo, QRHX decreased weight, improved blood lipid, relieved the degree of lipid deposition, reduced plaque area, decreased the levels of inflammatory cytokines (MCP-1, NLRP3, and TNFα), down-regulated the expression of iNOS, and up-regulated the expression of Arg-1. In vitro, QRHX down-regulated M1 markers, iNOS and CCR7, with lower concentrations of IL-1β; furthermore, QRHX up-regulated M2 markers, Arg-1, CD163, Ym-1, and Fizz-1, with higher concentrations of IL-4 and IL-10. RNA sequencing of both samples in vivo and in vitro suggested that NF-κB was the target pathway of QRHX to regulate macrophage polarisation; this result was validated at the gene and protein levels.QRHX induced M2 polarisation, reduced an inflammatory response, and played a role in stabilising plaque by mediating the NF-κB signalling pathway.Copyright © 2022. Published by Elsevier B.V.