Titanium dioxide nanoparticles (nano-TiO2) are usually applied to treat arsenic-polluted drinking water. Additionally, wide application of nano-TiO2 can enter into the environment inevitably. Furthermore, titanium dioxide nanoparticles (nano-TiO2) can adsorb ambient pollutants to modify their bioavailability in aquatic organisms, as well as their toxicity of its ambient pollutants. This study investigated arsenic accumulation, subcellular distribution and toxicity on two aquatic animals (Daphnia magna in freshwater and Artemia salina in salt water). Nano-TiO2 acts as a positive carrier, significantly facilitating D. magna’s ability to uptake As(V) as well as Artemia salina. In Artemia salina, As percentage in biologically active metal (BAM) fractions significantly decreased after the addition of 10 mg L−1 of nano-TiO2, while the fractional percentage of As increased in cellular debris. These lower As proportions in the sensitive fractions of cells indicate As(V) toxicity inhibition. Higher As(V) EC50 values in nauplii (by a magnitude from 1.97 to 2.76) compared to the control (nano-TiO2 free) as we increased nano-TiO2 concentrations from 1 to 1000 mgL−1, indicates that nano-TiO2 could alleviate As(V) toxicity in A. salina nauplii by enhancing efflux and decreasing the proportion of As in the sensitive fractions of cells. In contrast, in D. magna, even though As accumulation increased with increasing nano-TiO2 concentrations in D. magna, As(V) toxicity associated with nano-TiO2 exhibited a dual effect. Compared to the control, the increased As was mainly distributed in BDM (biologically detoxified metal), but Ti was mainly distributed in MSF (metal-sensitive fractions) with increasing nano-TiO2 levels. Differences in subcellular distribution demonstrated that adsorbed As(V) on nano-TiO2 could dissociate itself and be transported separately, which results in increased toxicity at higher nano-TiO2 concentrations. Decreased As(V) toxicity associated with lower nano-TiO2 concentrations results from unaffected As levels in MSFs (when compared to the control), where several As components continued to be adsorbed on nano-TiO2. Accordingly, arsenic toxicity on aquatic animals by nano-TiO2 depends on animal species and the behaviors of the co-contaminants inside, especially their assimilation in the digestive tract. More attention should be paid to the influence of nano-TiO2on As(V) assimilation in the digestive tract.