Stable isotope analysis provides a powerful tool for describing the energetic pathways in a variety of ecosystems. However, isotope ratios of animal tissues can be altered by preservation methods, potentially leading to biased estimates of energy pathways when they are not taken into account. Here, we investigated the direct preservation effects of formalin, ethanol, NaCl, and drying on the delta C-13 and delta N-15 of fish muscle tissues, as well as the ultimate effects on the reconstruction of the energy pathways. All preservation methods, except drying, had significant impacts on delta C-13 and delta N-15 values. The effects of preservation appear to be highly taxa-specific and no significant time-dependent variations in nearly 2-year duration of preservation. delta C-13 and delta N-15 values were generally changed dramatically within the early stage of the preservation process and became stable over a relatively long-term preservation. Using an isotopic balance mixing model, the isotope-based food web reconstruction reveals that, without preservation correction, the importance of the pelagic energetic pathways for the fishes could be misestimated, except for the drying preservation. These results highlight that preservation can bias the interpretation of food web reconstruction results.