Newcastle disease virus (NDV) is a significant enveloped virus within the Paramyxoviridae family, posing a major threat to the global poultry industry. Increasing evidence suggests that cholesterol-25-hydroxylase (CH25H) and its enzymatic product, 25-hydroxycholesterol (25HC), exhibit broad-spectrum antiviral activity properties by modulating lipid metabolism and various signaling pathways. However, the specific role of CH25H in regulating NDV infection and replication remains unclear. In this study, we demonstrate that CH25H significantly inhibits NDV replication by blocking viral entry through its enzymatic product, 25HC. Notably, a catalytic mutant of CH25H (CH25H-M), which lacks hydroxylase activity, still retains partial ability to inhibit NDV replication, suggesting the involvement of an enzyme-independent antiviral mechanism. Furthermore, we found that CH25H interacts with the viral nucleoprotein (NP), leading to a reduction in NP expression and inhibition of viral ribonucleoprotein (RNP) complex activity. These findings reveal that CH25H exerts antiviral effects through both enzyme-dependent and -independent mechanisms, providing new insights into its role in host defense and offering potential targets for the development of antiviral therapies.IMPORTANCECholesterol 25-hydroxylase (CH25H) is a multifunctional host protein that has been implicated in regulating the life cycles of various viruses. As a prototype of paramyxovirus, Newcastle disease virus (NDV) poses a significant threat to the global poultry industry, causing substantial economic losses. Uncovering the mechanisms of NDV-host interactions is crucial for unraveling the viral pathogenesis and the host immune response, which can drive the development of effective antiviral therapies. Here, we demonstrate that CH25H, whose expression is induced upon NDV infection, plays a pivotal role in restricting viral replication. Specifically, CH25H interacts with the viral NP and inhibits the viral RNP activity. These findings expand our understanding of CH25H's antiviral functions and offer new insights into virus-host interactions, providing potential targets for the development of novel antiviral drugs against NDV and related pathogens.