Aim To establish a single-step density gradient centrifugation method for isolating cholesterol-rich non-high density lipoprotein (CR-nHDL) particles and investigate the effect of oxidized CR-nHDL on foam cell formation. Methods A single-step density gradient centrifugation method using ioversol as the density medium was established to isolate CR-nHDL with a density of less than 1.008 g/mL from postprandial plasma of healthy volunteers. The density of the fraction was measured, and the cholesterol content was detected enzymatically. The lipoprotein content in the particles was analyzed by Western blot and proteomics. The isolated CR-nHDL was oxidized and co-cultured with mouse bone marrow-derived macrophages (BMDM), and its effect was compared with that of oxidized low density lipoprotein (ox-LDL). The ability of oxidized CR-nHDL to induce macrophage foam cell formation was assessed by measuring intracellular lipid accumulation. Results This method successfully isolated CR-nHDL with a density of less than 1.008 g/mL. Western blot results showed that this fraction contained apolipoprotein B100 (ApoB100) and apolipoprotein B48 (ApoB48) but not apolipoprotein AⅠ (ApoAⅠ). The results of proteomic analysis showed that there was no significant difference in the major apolipoprotein components between CR-nHDL prepared by ioversol method and low density lipoprotein (LDL) prepared by classical two-step method; lipidomics analysis results showed that the cholesterol ester content in the CR-nHDL prepared by the ioversol method was significantly higher than that in the LDL prepared by the classical method, which suggested that this method was more efficient in enriching cholesterol-containing lipoprotein particles, and the oxidized CR-nHDL obtained from this method could promote lipid accumulation in macrophage derived foam cells more than ox-LDL. Conclusion The single-step density gradient centrifugation of ioversol can quickly isolate CR-nHDL, and its oxidized form has a stronger ability to promote macrophage foam cell formation.