HIV fusion is mediated by the sequential interaction of the viral envelope glycoprotein with cellular receptors at the plasma membrane. We have previously reported that the upregulation of cellular ceramide levels following fenretinide treatment inhibits HIV fusion. As ceramide facilitates the internalization of a variety of microbes, we hypothesized that it may also promote the engulfment of HIV virions. Hence, we analyzed the effect of fenretinide treatment on virus binding and uptake. We observed that virus binding is not altered by fenretinide treatment. The distribution of HIV receptors was also unchanged. In contrast, virus uptake showed a significant increase. We have determined that fenretinide treatment promotes the internalization of virions from the plasma membrane and the accumulation of virus in the endocytic fraction of HeLa cells. This effect of fenretinide appears to be specific for virus as the endosomal accumulation of gp120, transferrin and horse-radish peroxidase was not increased. Notably, fenretinide increased the infectivity of influenza virus, which fuses in the endosomal compartment upon low pH activation. Our data suggest that fenretinide treatment effectively inhibits HIV infection by re-directing the virus to the endocytic pathway.