Nylon has been widely used all over the world, and most of it eventually enters the aquatic environment in the form of microplastics (MPs). However, the impact of Nylon MPs on aquatic ecosystem remains largely unknown. Thus, the long-term biological effects and toxicity mechanism of Nylon MPs on Microcystis aeruginosa (M. aeruginosa) were explored in this study. Results demonstrated that Nylon MPs had a dose-dependent growth inhibition of M. aeruginosa at the initial stage, and the maximum inhibition rate reached to 47.62% at the concentration of 100 mg/L. Meanwhile, Nylon MPs could obstruct photosynthesis electron transfer, reduce phycobiliproteins synthesis, destroy algal cell membrane, enhance the release of extracellular polymeric substances, and induce oxidative stress. Furthermore, transcriptomic analysis indicated that Nylon MPs dysregulated the expression of genes involved in tricarboxylic acid cycle, photosynthesis, photosynthesis-antenna proteins, oxidative phosphorylation, carbon fixation in photosynthetic organisms, and porphyrin and chlorophyll metabolism. According to the results of transcriptomic and biochemical analysis, the growth inhibition of M. aeruginosa is inferred to be regulated by three pathways: photosynthesis, oxidative stress, and energy metabolism. Our findings provide new insights into the toxicity mechanism of Nylon MPs on freshwater microalgae and valuable data for risk assessment of MPs.