The evolution of fluorescence is largely unexplored, despite the newfound occurrence of this phenomenon in a variety of organisms. We document that spiders fluoresce under ultraviolet illumination, and find that the expression of this trait varies greatly among taxa in this species-rich group. All spiders we examined possess fluorophores in their haemolymph, but bright fluorescence appears to result when a spider sequesters fluorophores in its setae or cuticle. By sampling widely across spider taxa, we determine that fluorescent expression is labile and has evolved multiple times. Moreover, examination of the excitation and emission properties of extracted fluorophores reveals that spiders possess multiple fluorophores and that these differ among some families, indicating that novel fluorophores have evolved during spider diversification. Because many spiders fluoresce in wavelengths visible to their predators and prey (birds and insects), we propose that natural selection imposed by predator-prey interactions may drive the evolution of fluorescence in spiders.