Cellular membrane cholesterol has been shown to support various membrane proteins. However, the role and function of membrane cholesterol in viral production are still unclear. Here, we investigated the effects of cholesterol depletion from the cell membrane on the production of hemagglutinating virus of Japan (HVJ; Sendai virus). Cholesterol depletion from LLC-MK2 cells by methyl-beta cyclodextrin treatment resulted in a marked increase in the production of both HVJ from the infected cells and virus-like particles from M-gene-transfected cells. The HVJ produced from cholesterol-depleted cells possessed a reduced amount of envelope cholesterol and showed a rather wide range of particle sizes and amount of envelope protein compared to HVJ produced from untreated cells. Direct depletion of envelope cholesterol from HVJ significantly impaired its infectivity, even without a change in envelope protein composition. These results suggest that membrane cholesterol plays important roles in regulating the production of infectious HVJ.