Only limited information is available on the effects of titanium dioxide nanoparticles (nano-TiO₂) on arsenite (As(III)) accumulation and its ensuing associated toxicity in aquatic organisms. This study characterized As(III) uptake, spatial and subcellular distribution, and efflux under different exposure treatments and its toxicity on Daphnia magna in the presence of nano-TiO₂. The results showed that the accumulated arsenic (As) content was significantly correlated to the corresponding accumulated titanium (Ti) content, implying that nano-TiO₂ as a carrier increased As(III) accumulation in D. magna. In addition, a significant spatial correlation between As and Ti accumulation further confirmed the “carrier” role of nano-TiO₂, while this role weakened under higher As(III) exposure levels due to its elevated toxicity and its limited adsorption capacity onto nano-TiO₂. Also, despite the increase in As(III) accumulation, nano-TiO₂ decreased D. magna toxicity. In particular, the 24 h As(III) EC₅₀ increased from 2.53 to 2.97 mg As per L, while nano-TiO₂ increased from 2 to 20 mg Ti per L. This reduction in toxicity resulted from the accumulation of most As in biologically detoxified metals (BDMs) and cellular debris as detoxified fractions. Moreover, the similarity in As and Ti subcellular distribution was clearly observed between 6 and 24 h exposure and between 75 μg As per L and EC₅₀ exposure levels of As(III). Therefore, regardless of the exposure time or exposure As(III) level, the subcellular distribution of As can itself explain its toxic effects on daphnids resulting from As(III) stress associated with nano-TiO₂. Interestingly, elevated nano-TiO₂ produced lower As efflux from daphnids, which showed that nano-TiO₂ still sequestered some As within this organism. Lastly, direct and reproduction efflux were the two main pathways that daphnids used to eliminate accumulated As and Ti. These findings can help us to better understand the ecological risks of nano-TiO₂ and its co-existing contaminants in the environment.