Carbon dioxide concentration and light conditions may greatly vary between mountainous and lowland areas determining the photosynthetic performance of plants species. This paper aimed to evaluate the photosynthetic responses of Lotus corniculatus, growing in a mountain and a lowland grassland, under low and high radiation and CO2 concentration. Net photosynthetic rate, stomatal conductance, transpiration rate, and intercellular CO2 concentration were measured while the water-use efficiency and the ratio of variable to maximal fluorescence were calculated. Photosynthetic response curves to different levels of radiation and intercellular CO2 partial pressure were estimated. Our results showed that high radiation and CO2 concentration enhanced water-use efficiency of plants at both sites, enabling them to use more efficiently the available water reserves under drought conditions. The increase of radiation and CO2 concentration would enhance the photosynthetic performance of the mountainous population of L. corniculatus, which overall seems to express higher phenotypic plasticity.