Display omitted •Cholesterol is essential for fusion of secretory vesicles and some enveloped viruses with cell membranes.•Cholesterol alters the distribution of SNARE and viral fusion proteins in membranes.•Cholesterol changes the penetration of fusion peptides in membranes.•Cholesterol alters the intrinsic membrane curvature and bending in membrane fusion.•Cholesterol alters the lifetime of hemifusion intermediates in membrane fusion. Cholesterol modulates the bilayer structure of biological membranes in multiple ways. It changes the fluidity, thickness, compressibility, water penetration and intrinsic curvature of lipid bilayers. In multi-component lipid mixtures, cholesterol induces phase separations, partitions selectively between different coexisting lipid phases, and causes integral membrane proteins to respond by changing conformation or redistribution in the membrane. But, which of these often overlapping properties are important for membrane fusion?—Here we review a range of recent experiments that elucidate the multiple roles that cholesterol plays in SNARE-mediated and viral envelope glycoprotein-mediated membrane fusion.