Marsilea quadrifolia, an amphibious fern, has the ability to develop heterophyllous, aerial and submerged leaves. In contrast to submerged leaves, aerial leaves have trichomes on both surfaces. To examine if the presence of trichomes can reflect excess light and hence reduce the risk of being damaged by excess light, we compared the optical properties and chlorophyll a fluorescence, in particular the maximum PSII photochemical efficiency (Fv/Fm), of M. quadrifolia leaves with trichomes (intact) and those having trichomes removed (de-trichomed). Photosynthetic gas exchange measurements were also conducted to quantify transpirational water loss and instantaneous water use efficiency (WUE) of M. quadrifolia intact and de-trichomed leaves. The results showed that removal of trichomes neither affected the optical properties in the visible part of the solar spectrum nor the midday depression of Fv/Fm values of leaflets. In contrast, significantly increased in transpiration rates and decreased rates in WUE were found in de-trichomed leaflets in comparison to intact ones. These results imply that the presence of trichomes is of more importance in reducing water loss than in reflecting light and protecting M. quadrifolia against the potentially damaging effect of photoinhibition in aerial environments.