Influenza is an RNA virus encapsulated in a lipid bilayer derived from the host cell plasma membrane. Previous studies showed that influenza infection depends on cellular lipids including the sphingolipids sphingomyelin and sphingosine. Here we examined the role of a third sphingolipid, glucosylceramide, in influenza infection following CRISPR/Cas9-mediated knockout of its metabolizing enzyme glucosylceramidase (GBA). After confirming GBA knockout of HEK 293 and A549 cells by both western blotting and lipid mass spectrometry, we observed diminished infection in both KO cell lines by a PR8 (H1N1) GFP reporter virus. We further showed that reduction in infection correlated with impaired influenza trafficking to late endosomes, and hence fusion, and entry. To examine whether GBA is required for other enveloped viruses, we compared entry mediated by the glycoproteins of Ebola, influenza, vesicular stomatitis, and measles viruses in GBA knockout cells. Entry inhibition was relatively robust for Ebola and influenza, modest for VSV, and mild for measles, suggesting a greater role for viruses that enter cells by fusing with late endosomes. As the virus studies suggested a general role for GBA along the endocytic pathway, we tested and found that trafficking of epidermal growth factor to late endosomes, as well as degradation of its receptor, were impaired in GBA knockout cells. Collectively our findings suggest that GBA is critically important for endocytic trafficking of viruses as well as cellular cargos including growth factor receptors. Modulation of glucosylceramide levels may therefore represent a novel accompaniment to strategies to antagonize ´late penetrating´ viruses, including influenza.IMPORTANCE Influenza is a viral pathogen responsible for the second largest pandemic in human history. A better understanding of how influenza enters host cells may lead to more efficacious therapies against emerging strains of the virus. Here we show that the glycosphingolipid metabolizing enzyme glucosylceramidase is required for optimal influenza trafficking to late endosomes and consequent fusion, entry, and infection. We also provide evidence that promotion of influenza entry by glucosylceramidase extends to other endosome-entering viruses and is due to a general requirement for this enzyme, and hence optimal levels of glucosylceramide, for efficient trafficking of endogenous cargos such as the EGF receptor, along the endocytic pathway. This work therefore has implications for the basic process of endocytosis as well as pathogenic processes including virus entry and Gaucher disease.