Cholesterol-rich lipid rafts have been shown to play important roles in the life cycle of various non-enveloped and enveloped viruses. Deletion of cholesterol from lipid rafts could influence different steps of viral replication cycle including entry, infection, assembly and release. Caprine parainfluenza virus type3 (CPIV3) is a newly identified member of Paramyxoviridae family. CPIV3 is highly prevalence and threatened the goat industry in China. The infection mechanism of CPIV3 is under exploring and still not fully understood, the roles of cholesterol and lipid rafts for CPIV3 infection remains unclear. In this study, we investigated the association of cholesterol and lipid rafts with CPIV3 during the different viral replication stages (binding, entry and infection) in two cells [MDBK and goat bronchial epithelial (GBE) cells]. Methyl-β- cyclodextrin (MβCD) was used to deplete cholesterol from cell and viral membranes. The results showed that MβCD treatment significantly inhibited CPIV3 entry and infection in these two cells with a dose-dependent manner, but didn't impair the binding of CPIV3. Addition of exogenous cholesterol to the cells after MβCD treatment restored the viral infection. In addition, treatment of MβCD only before virus-entry showed inhibitory effect in MDBK cells. Depletion of cholesterol from virion envelop also decreased the entry and infection of CPIV3 in the two cells. Furthermore, lipid rafts isolation test indicated that viral proteins (HN and N) co-localized with lipid rafts during infection in MDBK and GBE cells. Viral N protein co-localized with caveolin-1 (the marker of lipid rafts) in these two cells both at the entry and infection steps, as detected by con-focal laser scanning microscopy test. In conclusion, the results presented here demonstrated that cholesterol rich lipid rafts play an important role in CPIV3 life cycle. The findings give new insights on understanding of the mechanism of CPIV3 infection and provide a new anti-CPIV3 strategy.