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The secretion, accumulation, and composition of extracellular polymeric substances (EPS) are pivotal factors influencing microalgal growth as well as wastewater recycling. Until now, the accumulation and inhibition mechanism of EPS of Chlorella vulgaris during cycling cultivation is not fully understood. The purpose of this study was to explore how different light qualities regulate the secretion, chemical composition, and structure of microalgal EPS, and subsequently influence the recycling of culture wastewater. After four cycles of cultivation, C. vulgaris under green light produced the highest EPS production and lowest biomass production, which were 82% higher and 17% lower, respectively, compared to white light, which yielded the least EPS production and the highest biomass production. EPS under different light qualities all exhibited a fibrillar structure with a sheet-like surface, but differed in composition. Compared with the other groups, EPS under green light showed a significant increase in polysaccharides, proteins, and humic acid-like compounds, as well as an increased proportion of arabinose and rhamnose, according to monosaccharide composition analysis. Transcriptome analysis indicated that the up-regulation of metabolic pathways linked to glycolysis/gluconeogenesis, TCA cycle, lipid synthesis, and ABC transporters promoted EPS accumulation. Additionally, EPS could target light-harvesting complex (LHC) and electron transport chain, down-regulating the photosynthetic pathway, which ultimately inhibited microalgal growth under green light. This study provides a theoretical foundation for the light regulation and circulation culture of microalgae, as well as for microalgal wastewater treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)