Aquatic plants are vital components of shallow aquatic ecosystems, and they can substantially contribute to food webs. However, the large spatial and temporal variations of δ13C and δ15N signatures of aquatic plants have hindered the interpretation of their trophic interactions with organisms at higher trophic levels, and the effects of temperature on plant isotopic signatures remain to be fully elucidated. Herein, we cultured three common submerged macrophytes [Elodea nuttallii (Planch.) St. John, Vallisneria spiralis L., and Potamogeton lucens L.] at four temperatures (10, 15, 20 and 25 °C) for 16 weeks and analyzed their δ13C and δ15N signatures. Results showed that temperature altered the isotopic signatures of all three plant species. δ13C and δ15N varied by 16.06‰ and 11.68‰ in P. lucens at different temperatures, respectively. Plant δ15N significantly decreased with rising temperature in all three plant species and was correlated with plant growth, N content, and pore water dissolved inorganic N (DIN) concentrations. Conversely, δ13C responded non-linearly with temperature: a hump-shaped response of δ13C with temperature was observed for P. lucens. Plant δ13C was not correlated with any of the measured parameters. Temperature can alter plant metabolism and photosynthesis and the compositions and concentrations of C and N sources, thereby influencing plant δ13C and δ15N signatures, respectively. Temperature plays a key role in altering plant C and N isotopic signatures. Therefore, we recommend future studies to carefully consider the effects of temperature on plant stable isotopic signatures when interpreting the food contribution of aquatic plants in food webs and long-term environmental changes via historical isotopic signatures of plants exposed to different temperatures, particularly in light of changing climate conditions.