Emerging evidence indicates that in addition to native pentameric C-reactive protein (CRP), monomeric CRP (mCRP) also plays an active role in inflammation associated with cardiovascular diseases. mCRP activates endothelial cells, one of the critical events in cardiovascular diseases; however, the underlying molecular mechanisms are incompletely understood. Here we report that association of mCRP with human aortic and coronary artery endothelial cells is predominantly due to membrane insertion rather than binding to the surface proteins Fc gammaRs and proteoglycans. We identify lipid rafts as the preferential membrane microdomains for mCRP anchorage. mCRP binding depends on membrane cholesterol content and is synergistically mediated by the putative cholesterol binding consensus sequence of CRP (aa 35-47) and the C-terminal octapeptide (aa 199-206). Conversely, disrupting lipid rafts with methyl-beta cyclodextrin or nystatin abrogated mCRP-induced cytokine release, reactive oxygen species generation, and adhesion molecule expression in endothelial cells. Furthermore, ex vivo treatment of rabbit thoracic aorta and carotid artery segments with nystatin prevented mCRP-induced IL-8 release. Our data identify mCRP-lipid raft interaction as an important mechanism in mediating cellular responses to mCRP and lend further support to the notion of mCRP regulation of endothelial cell function during inflammation.