The study identifies the cholesterol transporter Niemann-Pick C1 (NPC1) as a critical host factor required for Ebola virus (EboV) and Marburg virus (MarV) entry into human cells. A genome-wide haploid genetic screen in human cells revealed that mutations disrupting all six members of the HOPS multisubunit tethering complex and NPC1 were enriched in cells resistant to infection by EboV. These mutations blocked viral membrane fusion and escape from the endosomal/lysosomal compartment, independent of NPC1's known role in cholesterol transport. NPC1-deficient primary fibroblasts derived from Niemann-Pick type C1 disease patients were resistant to EboV and MarV infection, but remained susceptible to other viruses. Treatment of cells with the cholesterol synthesis inhibitor U18666A also blocked EboV and MarV infection. These findings suggest that NPC1 plays a unique role in filovirus entry and may provide a target for antiviral therapy.The study identifies the cholesterol transporter Niemann-Pick C1 (NPC1) as a critical host factor required for Ebola virus (EboV) and Marburg virus (MarV) entry into human cells. A genome-wide haploid genetic screen in human cells revealed that mutations disrupting all six members of the HOPS multisubunit tethering complex and NPC1 were enriched in cells resistant to infection by EboV. These mutations blocked viral membrane fusion and escape from the endosomal/lysosomal compartment, independent of NPC1's known role in cholesterol transport. NPC1-deficient primary fibroblasts derived from Niemann-Pick type C1 disease patients were resistant to EboV and MarV infection, but remained susceptible to other viruses. Treatment of cells with the cholesterol synthesis inhibitor U18666A also blocked EboV and MarV infection. These findings suggest that NPC1 plays a unique role in filovirus entry and may provide a target for antiviral therapy.