INTRODUCTION: High-density lipoprotein (HDL) serves as a key protective factor against elevated low-density lipoprotein cholesterol (LDL-C) levels in patients with familial hypercholesterolemia (FH). This study aimed to assess whether HDL's ability to acquire free cholesterol (FC) from triglyceride-rich lipoproteins (TGRLs) during lipolysis, as a part of reverse remnant-cholesterol transport (RRT) pathway, is impaired in FH patients. METHODS: This case-control study included 16 homozygous (HOFH) and 18 heterozygous (HEFH) patients with FH, along with 20 healthy controls. The free cholesterol transfer assay measured the ability of HDL (as apoB-depleted serum) to uptake fluorescently labelled free cholesterol (TopF) from TGRLs during lipoprotein lipase (LPL)-mediated lipolysis. RESULTS: No significant difference was observed among the groups in the overall capacity of HDL to acquire FC from TGRLs during LPL-mediated lipolysis. When normalizing for LDL-C and non-HDL-C levels, significantly lower TopF transfer/LDL-C and TopF transfer/non-HDL-C ratios were observed in HOFH patients in comparison with HEFH and healthy subjects. Lower TopF transfer/LDL-C (OR: 0.605, 95% CI: 0.428-0.857; p < 0.01) and TopF transfer/non-HDL-C (OR: 0.539, 95% CI: 0.339-0.859; p < 0.01) ratios were independently associated with HOFH, while, after adjusting for age only the former association remained significant (OR: 0.646, 95% CI: 0.430-0.971). CONCLUSION: Although HDL's direct capacity to acquire free cholesterol from TGRLs did not differ significantly between the groups, the reduced TopF transfer/LDL-C and TopF transfer non-HDL-C ratios in FH patients is consistent with impaired HDL function. These ratios may serve as a potential biomarker for identifying high-risk FH phenotypes, warranting validation in larger, prospective studies.