Productive viral infection begins with attachment to a susceptible cell, and viruses have evolved complex mechanisms to attach to and subsequently enter cells. Prior to engagement with a cellular receptor, viruses frequently interact with nonspecific attachment factors that can facilitate virus-receptor interactions and viral entry. Polyamines, small positively-charged molecules abundant in mammalian cells, mediate viral attachment, though the mechanism was not fully understood. Using the Coxsackievirus B3 (CVB3) enterovirus model system, we show that polyamines mediate viral attachment both directly and indirectly. The polyamine putrescine specifically enhances viral attachment to cells depleted of polyamines. Putrescine’s positive charge mediates its ability to enhance viral attachment, and polyamine analogs are less efficient at mediating viral attachment. In addition to this direct role of polyamines in attachment, polyamines facilitate the cellular expression of heparan sulfates, negatively-charged molecules found on the cell surface. In polyamine-depleted cells, heparan sulfates are depleted from the surface of cells, resulting in reduced viral attachment. We find that this is due to polyamines’ role in the process of hypusination of eukaryotic initiation factor 5A, which facilitates cellular translation. These data highlight the important role of polyamines in mediating cellular attachment, as well as their function in facilitating cellular heparan sulfate synthesis.