An investigation into the influence of UV-irradiation on regenerated silk fibroin dissolved in water was carried out using UV–Vis and fluorescence spectroscopy. It was found that the absorption of regenerated silk fibroin in solution increased during UV-irradiation of the sample, most notably between 250 and 400 nm. Moreover, after UV-irradiation a wide peak emerged between 290 and 340 nm with maximum at about 305 nm. The new peak suggests that new photoproducts are formed during UV-irradiation of regenerated silk fibroin. The fluorescence of regenerated silk fibroin was observed at 305 nm, at 480 nm and at 601 nm after excitation at 275 nm. UV-irradiation caused fluorescence fading at 305 nm and at 601 nm. The increase of fluorescence was observed at 480 nm, probably due to formation of new photoproducts. After excitation at 305 nm the fluorescence of regenerated silk fibroin was observed at 340 nm and at 400 nm. UV-irradiation caused fluorescence fading at 340 nm. FTIR spectroscopy showed that primary structure of regenerated silk fibroin was not significantly affected by UV radiation. SDS-PAGE chromatography showed alterations of molecular weight of silk after UV exposure.