Exocytosis is a fundamental cellular process, pivotal in a wide range of cell types, used to deliver chemical messengers from one cell to another cell or tissue. While a tremendous amount of knowledge has been gained in the past several decades about the exocytotic machinery, recently it has become clear that the role of membrane lipids is also crucial in this process. In particular, the critical role of the abundant and ubiquitous cholesterol molecules has not been well-defined. Early insight has been gleaned from single cell amperometric studies on several commonly used secretory cell models, including chromaffin cells and PC12 cells; however, these secretory cell models are not ideal because manipulations of membrane cholesterol content may influence downstream cholesterol-dependent processes, making data interpretation difficult. Herein, blood platelets are employed as a simpler secretory cell model based on their anuclear nature and unique chemical messenger exocytosis behavior. Carbon-fiber microelectrochemistry was employed to measure real-time exocytosis from single platelets with depleted or enriched cholesterol either in the naturally occurring form or as the synthetic analogue epicholesterol. The experimental results show that membrane cholesterol directly modulates the secretion efficiency of individual platelets, as well as the kinetics of secretion events. Moreover, substitution of platelet membrane cholesterol with epicholesterol yields exocytotic behavior indistinguishable from that of normal platelets, arguing against the possibility of cholesterol-specific interactions in regulating exocytosis. It is clear from this work that membrane cholesterol plays a critical biophysical, rather than biochemical, role in platelet exocytosis and likely in exocytosis in general.