Alphaviruses are enveloped viruses that infect host cells by receptor-mediated endocytosis. The subsequent envelope fusion to cell membrane is triggered by low pH and requires the presence of both cholesterol and sphingolipids in the target membrane. In this study, we have examined the role of cholesterol in the Mayaro virus envelope, using methyl-β-cyclodextrin depletion. The pretreatment of virions with methyl-β-cyclodextrin efficiently depleted envelope cholesterol in a dose-dependent manner and significantly reduced Mayaro virus infectivity. Despite of the requirement of cholesterol for alphaviruses membrane fusion reaction, cholesterol depletion from either cellular plasma membrane or virus envelope seems to increase fusion event, as accessed by spectrofluorometric analysis. Transmission electron microscopy analysis revealed that envelope cholesterol depletion markedly affected virus morphology, by permeabilization of the viral membrane, suggesting that this lipid plays a role in the maintenance of viral particle integrity. We also investigate the effect of cellular cholesterol depletion before and after infection on virus infectivity, confirming the requirement of this lipid for both entry and exit steps of viral life-cycle. Our results suggest that cholesterol present in Mayaro virus is a key component for virus entry and in the maintenance of viral structure.