Stable isotope analysis (SIA) provides essential information step toward a better understanding of trophic ecology. However, the interpretation of SIA results relies on assumptions about the trophic discrimination factor (TDF) that aims to improve the accuracy but may lead to bias. In aquatic ecosystems, most biota are poikilothermic organisms, thus temperature is one of the most important parameters affecting all biological processes, including trophic discrimination of stable isotopes. Therefore, we conducted an experiment to establish TDF for freshwater fish under different temperature regimes (15, 25 °C, and natural ambient pond temperatures from July to September). We used common carp ( Cyprinus carpio, L., 1758) as a model organism. In the first phase of the experiment (weeks 1-6), all fish were fed by a defined fish feed to establish a baseline isotopic signal for all individuals. In the second phase (weeks 7-18), fish were randomly divided into three temperature groups and were all fed by different fish feed. Results indicated temperature dependent TDF, whereby nitrogen showed higher temperature dependency, where highest TDF were observed in 15 °C group and lowest in 25 °C group. In than carbon, where equilibrium of muscle isotopic value of was reached only in the natural, ambient temperature regime. Presumably, discrimination of nitrogen isotopes was related to metabolic turnover rates, where all values were significant, while in carbon only significant values was found in natural, ambient temperature regime These findings highlight the importance of considering environmental thermal conditions in evaluating stable isotope signatures in trophic studies.