Segestria florentina (Rossi 1790) (Segestriidae) displays iridescent green coloration on the paturons of the chelicerae. This was confirmed by reflectance measurements, which gave a spectral peak at 505 nm. Scanning electron microscopy did not identify cuticular surface scales or sculpturing, suggesting that the cause of the iridescence was subsurface. Transmission electron microscopy revealed 86 alternate dark and light layers in the exocuticle, the mean dimensions of which were 126 nm ± 28 nm and 88 nm ± 55 nm respectively. The identity of each layer was initially unclear. However, by using a combination of materials with different refractive indices in calculations of theoretical reflectance spectra, we concluded that they were most likely to be composed of chitin and air, since a peak of 480 nm was obtained, which most closely matched that which was recorded. The function of the green color is not clear, since S. florentina has relatively poor vision and relies predominantly on vibratory and acoustic signals. The study provides useful information relevant to research into the evolution of structural colors in spiders and, more generally, in nature.