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Povidone iodine (PVP‐I) is widely used as an efficient antiseptic disinfectant, which can kill various pernicious microbe. PVP‐I also exhibits certain toxic effects to the environment. To study the comprehensive toxic effects of PVP‐I on microalgae, different concentrations including 0, 7, 14, 28, 56 and 112 mg/L were set. The effects of PVP‐I on the growth performance, microstructure, photosynthesis, antioxidant levels and photosynthesis associated genes expressions of microalgae were investigated. The results showed that PVP‐I had significant effects on the growth and physiological functions of C. pyrenoidosa. Under the observation of scanning electron microscope, PVP‐I of 56 and 112 mg/L could destroy the cytoderm of C. pyrenoidosa directly. Under the PVP‐I exposure, the activity of SOD and CAT fluctuated up and down, while MDA content continued to rise. The relative gene expression of PsbA gene was significantly up‐regulated in high concentration of 112 mg/L PVP‐I. The relative gene expression of rbcL and rbcS gene were upregulated in 28 mg/L PVP‐I. The results of present study showed that PVP‐I could induce the production of oxidative stress response in C. pyrenoidosa, inhibit the expression of genes related to photosynthesis of C. pyrenoidosa and affect the photosynthesis of C. pyrenoidosa. The present study will provide a reference for the safe application of PVP‐I. [ABSTRACT FROM AUTHOR]