Aim To investigate the role of cholesterol in the regulation of lipid raft and the function of platelets. Methods Using in vitro incubation of methyl β-cyclodextrin (MβCD) and in vivo elevation of peripheral blood total cholesterol levels to remove and load platelet cholesterol, respectively. Cholera toxin B staining combined with flow cytometry was used to detect platelet lipid raft content, fluorescence antibody staining combined with flow cytometry was used to detect the expression levels of P-selectin and activated integrin αⅡbβ3, annexin V labeling combined with flow cytometry was used to detect the level of phospholipid efflux, in vitro experimental system and rat tail bleeding experiment were used to detect platelet aggregation ability. Results The content of lipid raft on B lymphocytes decreased with the removal of cholesterol, while in vitro incubation of MβCD to remove platelet cholesterol significantly increased its lipid raft level (P＜0.05). Consistent with this, in vivo cholesterol loading increased the lipid raft content of B lymphocytes but decreased the lipid raft content of platelets (P＜0.05). The increase in lipid raft after removing cholesterol was not conducive to platelet activation and aggregation function. In vivo cholesterol loading downregulated platelet lipid raft content (P＜0.05), enhanced its ability to respond to low concentration stimulant for activation aggregation and coagulation, and this enhancing effect disappeared after cholesterol removal. Conclusion Platelet cholesterol is a key regulator of platelet lipid raft content and platelet function, which can negatively regulate lipid raft, promote platelet activation, and enhance their coagulation function.