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Combined pollutions of arsenic (As) and copper (Cu) are common in water bodies near mines, non-ferrous metal smelting and power plants. This study investigated the effect of Cu(II) on the absorption and speciation of As(V) by microalgae. We compared the absorption and speciation of arsenic by microalgae (mainly Cyanophyta and Chlorophyta) when exposed to single As(V) with that exposed to As-Cu co-exposure in laboratory. The results showed that in the case of single As(V) exposure, the inhibitory effect of As(V) on microalgae was primarily affected by the exposure time, instead of the concentration of As(V) in the water solution. Compared with single As(V) exposure, the presence of Cu(II) under As-Cu co-exposure promoted the absorption and accumulation of As(V) by algae. The combination effect of As and Cu on algae was antagonistic instead of synergistic within the tolerance range of algae to them. In the presence of Cu(II), more monomethylarsonous acid (MMA) and dimethylarsinous acid (DMA), which are volatile organic arsenic compounds, were produced in algae compared with the control. The finding that Cu(II) can mediate the absorption and speciation processes of arsenic in algae has significance in possible bioremediation of arsenic pollution in aquatic environment.