Recently, there has been increasing global concerns about the ecological impact of micro- and nano-sized polystyrene particles in marine environments. However, the potential threats of polystyrene nanoparticles to primary producers in freshwater systems remain unclear. Here, we explore the metabolite profiles and signaling pathways of freshwater cyanobacterium Synechococcus elongatusin response to short-term (48 h) amino-modified polystyrene nanoparticle (PS-NH2; 50 nm) exposure. We demonstrate that the positively charged PS-NH2were acutely toxic to S. elongatuswith an EC50of 3.81 μg mL−1. Non-targeted metabolomics analyses suggested oxidative stress and membrane destruction as the main causes of PS-NH2toxicity. Furthermore, disruption of glutathione metabolism and damage to membrane integrity were confirmed using two engineered strains of S. elongatus. These results provide new insights into polystyrene nanoparticles' impact on primary producers, improving the understanding of the toxic mechanisms of polystyrene nanoparticles in the freshwater primary producers.