Nano titanium dioxide (nTiO2) generally shows low toxicity to organisms under light-emitting diode (LED) light. However, nTiO2 can induce production of reactive oxygen species (ROS) under ultraviolet (UV) light due to its photocatalytic activity. Therefore, it is reasonable to expect the enhancement of nTiO2 toxicity under sunlight. To test this hypothesis, we compared the toxicity of nTiO2 to Brachionus calyciflorus under simulated sunlight and LED light. The results showed that the 24 h-LC50 of nTiO2 to B. calyciflorus under LED light and simulated sunlight were 24.32 (95% CI: 14.54–46.81 mg/L) and 10.44 mg/L (95% CI: 6.74–17.09 mg/L), respectively. Compared with the blank control, treatments with nTiO2 significantly affected life-table demographic parameters, population growth parameters and swimming linear speed under both simulated sunlight and LED light. However, life expectancy, net reproductive rate, average lifespan, maximal population density, and swimming linear speed in the treatments of nTiO2 at 0.1, 1, and/or 10 mg/L showed markedly lower values under simulated sunlight than those under LED light, suggesting that simulated sunlight could enhance the toxicity of nTiO2. In addition, markedly higher catalase (CAT) activity and malondialdehyde (MDA) content but lower glutathione (GSH) content were observed in treatment with 10 mg/L nTiO2 under simulated sunlight than that under LED light. The results showed that compared with LED light, simulated sunlight significantly induced more oxidative stress in the presence of nTiO2, and the ROS production was mainly localized to the corona and digestive tract of rotifers by confocal laser scanning microscope. Exposure to 10–50 μM of vitamin C, that is an effective ROS scavenger, could rescue the swimming linear speed of rotifers to the normal level in the blank control. These results suggested that oxidative damages on cell membrane might be the vital mechanism underlying the toxicity enhancement of nTiO2 to rotifers under simulated sunlight. Thus, the previous publications under LED light may underestimate the real toxicity and environmental risk of nTiO2 in natural conditions.