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Microalgae-based bioremediation presents an alternative to traditional biological wastewater treatment. However, its efficiency is still challenging due to low microalgal activities and growth rate in wastewater. Iron plays an important role in microbial metabolism and is effective to stimulate microbial growth. In this study, a novel approach was proposed to simultaneously promote microalgal activity and nutrient uptake from wastewater using nanoscale zerovalent iron (nZVI), and the underlying molecular mechanism was explored. Compared to the control, 0.05 mg/L of nZVI significantly enhanced biomass production by 113.3% as well as NH4 +-N and PO4 3–-P uptake rates by 32.2% and 75.0%, respectively. These observations were attributed to the enhanced metabolic pathways and intracellular regulations. Specifically, nZVI alleviated the cellular oxidative stress via decreased peroxisome biogenesis as indicated by reduced reactive oxygen species, enzymes, and genes involved. nZVI promoted ammonium assimilation, phosphate metabolism, carbon fixation, and energy generation. Moreover, nZVI regulated the biosynthesis and conversions of intracellular biocomposition, leading to increased carotenoid, carbohydrate, and lipid productions and decreased protein and fatty acid yields. The above metabolisms were supported by the regulations of differentially expressed genes involved. This study provided an nZVI-based approach and molecular mechanism for enhancing microalgal activities and nutrient uptake from wastewater.