Microbial extracellular polymeric substances (EPS) widely exist in natural environments and affect the migration and transformation of pollutants in aquatic environments. Previous works report that EPS have some reducing functional groups and can reduce heavy metals. However, because of the weak reducing capability of EPS, the reduction of heavy metals by EPS without cells is extremely slow, and its effect on heavy metals species is insignificant. In this work, the accelerated reduction of silver ions (Ag+) by EPS from Shewanella oneidensis MR-1 under illumination was investigated. UV–visible spectroscopy, transmission electron microscopy (TEM) coupled with an energy dispersive spectrometer (EDS) and X-ray photoelectron spectroscopy (XPS) were used to confirm the formation of silver nanoparticles (AgNPs) via the reduction of Ag+ by EPS under light illumination. The Ag+ reduction by EPS follows pseudo-first-order kinetics under both visible and UV light, and the light irradiation can significantly accelerate AgNPs formation. On the one hand, visible light can excite AgNPs for their surface plasma resonance (SPR) and accelerate the electrons from the EPS to adjacent Ag+. On the other hand, EPS molecules may be excited by UV light to produce strong reducing species, which enhance Ag+ reduction. Moreover, pH, dissolved oxygen were found to affect the formation of AgNPs by EPS. This work proves the reducing capability of EPS on the reduction of Ag+, and this process can be accelerated under light illumination, which may affect the speciation and transformation of heavy metals in natural waters. Display omitted •AgNPs formation via reduction of Ag+ by EPS under illumination was confirmed.•Light illumination can significantly enhance the reduction of Ag+ by EPS.•The Ag+ reduction by EPS follows pseudo-first-order kinetics.•The mechanism of effect of light on the reduction of Ag+ by EPS was proposed.•pH and dissolved oxygen will influence the reduction of Ag+ by EPS.