Titanium dioxide nanoparticles (TiO2 NPs) have raised concern of environmental risks due to their widespread applications, but little is known about the potential toxicity of TiO2 NPs to aquatic plants. The aim of this work was to study the effects of TiO2 NPs on Lemna minor and to study the behavior of TiO2 NPs under modified ISO 20079 test conditions. TiO2 NPs had a tendency to aggregate in ISO (Steinberg) growth medium, but modification of the standard growth medium enabled the exposure of L. minor to TiO2 NPs. By dilution of the growth medium (1:10), and exposure under semi-static conditions with medium renewal every second or third day, the size of TiO2 particles remained rather stable throughout the test period. TiO2 NPs showed no adverse effect on the growth rate or chlorophyll a content of L. minor, even at a high exposure concentration of 5mgL−1 and extended exposure time of 14 days. TiO2 NPs attached onto L. minor cell walls, but no cellular uptake was observed. Although TiO2 NPs were not toxic to L. minor, the potential transfer of TiO2 NPs in aquatic food chains, e.g. attached to the plant leaves and other biological surfaces may be of importance, causing exposure of other organisms and contributing to the environmental fate of nanoparticles.