Abstract One target of development and application of TiO 2 nanoparticles (nano‐TiO 2 ) is photochemical degradation of contaminants and photo‐killing of microbes and fouling organisms. However, few ecotoxicological studies have focused on this aspect of nano‐TiO 2 , specifically whether this photoreactivity might significantly increase hazard and risk of the materials in the natural environment. In the present study, we evaluated acute phototoxicity of nano‐TiO 2 under simulated solar radiation (SSR) to two aquatic species– Daphnia magna and Japanese medaka, using 48‐h and 96‐h assays, respectively. A thorough characterization of the exposure system was performed by measuring particle agglomeration and TiO 2 concentration in suspension in a time‐course manner. Sedimentation and loss of bulk concentration of nano‐TiO 2 particles occurred at all concentrations above 2 mg/L and was more significant as concentration increased. Phototoxicity of nano‐TiO 2 under SSR was enhanced by two to four orders of magnitude as compared to toxicity under ambient laboratory light, with a 48‐h median lethal concentration (LC50) of 29.8 µg/L in D. magna and a 96‐h LC50 of 2.2 mg/L in medaka. Our results also indicate that these effects are dependent on simultaneous exposure of the organisms to nanoparticles and SSR. This dramatic increase in toxicity of nano‐TiO 2 at environmentally realistic levels of SSR indicates the need to incorporate this mode of action into risk assessment for nano‐TiO 2 and other photoreactive nanomaterials. Environ. Toxicol. Chem. 2012; 31: 1621–1629. © 2012 SETAC