The rapid growth in silver nanoparticles (AgNPs) commercialization has increased environmental exposure, including aquatic ecosystem. It has been reported that the AgNPs have damaging effects on photosynthesis and induce oxidative stress, but the toxic mechanism of AgNPs is still a matter of debate. In the present study, on the model aquatic higher plant Spirodela polyrhiza, we found that AgNPs affect photosynthesis and significantly inhibit Photosystem II (PSII) maximum quantum yield (F-v/F-m) and effective quantum yield (phi(PSII)). The changes of non-photochemical fluorescence quenching (NPQ), light-induced non-photochemical fluorescence quenching [Y(NPQ)] and non-light-induced non-photochemical fluorescence quenching [Y(NO)] showed that AgNPs inhibit the photo-protective capacity of PSII. AgNPs induce reactive oxygen species (ROS) that are mainly produced in the chloroplast. The activity of ribulose-1, 5-bisphosphate carboxylase-oxygenase (Rubisco) was also very sensitive to AgNPs. The internalized Ag, regardless of whether the exposure was Ag(+)or AgNPs had the same capacity to generate ROS. Our results support the hypothesis that intra-cellular AgNP dissociate into high toxic Ag+. Rubisco inhibition leads to slowing down of CO2 assimilation. Consequently, the solar energy consumption decreases and then the excess excitation energy promotes ROS generation in chloroplast.