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Enveloped virus invasion relies on spike glycoprotein-mediated membrane fusion. Cholesterol that serves crucial roles in modulating protein conformations and membrane properties, plays an essential role in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cell entry. However, the precise regulatory mechanism of cholesterol in SARS-CoV-2 fusion remains unknown. Here, using an in vitro vesicle-vesicle content mixing assay, we demonstrated that the addition of cholesterol enhanced SARS-CoV-2 spike-mediated vesicle-vesicle fusion, with this enhancement being dependent on the C-terminal cytoplasmic domain of spike. Further single-vesicle analyses demonstrate this enhancement primarily stems from increased docking probability, with cholesterol exerting mild effect on fusion probabilities. In the cell-based membrane fusion assay, cholesterol depletion from spike containing membrane significantly reduces syncytia formation and SARS-CoV-2 pseudovirus infection, indicating its modulatory role in this process. Using structured illumination microscopy (SIM) based super-resolution imaging and single-molecule photobleaching microscopy, we demonstrated that spike proteins tended to form into an oligomeric cluster in the presence of cholesterol, likely through the interaction between cholesterol and palmitoylated cysteine rich region (CRR) in the C-terminus of spike. Last, substitution of residues of CRR with alanine in the C-terminus of spike abolished both the cholesterol-induced spike clustering and the cholesterol-dependent enhancement of vesicle docking. Taken together, our results suggest that cholesterol may induce the oligomerization of spike through specific interactions with its CRR, with this structural clustering critically mediating viral docking to host cell membranes, thereby promoting the subsequent membrane fusion and viral entry processes.