Lipid rafts are special microdomains in the plasma membrane. They are enriched in sphingolipids and cholesterol, playing critical roles in many biological processes. The purpose of this study is to analyze the requirement of cholesterol, a crucial component of lipid rafts for cell infection by pseudorabies virus (PrV). Cholesterol of plasma membrane or viral envelope was depleted with methyl-beta-cyclodextrin (MβCD), and the infectivity of three strains of PrV was determined with plaque assays. The effect of adding cholesterol to MβCD-treated cells and viruses on cell infection was analyzed. Furthermore, effect of post-adsorption cholesterol depletion on PrV infection was investigated. We show that cholesterol depletion of either the plasma membrane or the viral membrane by MβCD significantly impaired the infectivity of PrV strains Kaplan, Becker, and Bartha K-61. The virus was shown to have lower cholesterol content and to respond to lower MβCD concentrations. Exogenous cholesterol added to either MβCD-treated cells or virions partially restored the virus infectivity. Optimal PrV infection requires cholesterol in viral and plasma membranes.