Stable isotope analysis (SIA) has become an important tool to investigate diet shift, habitat use and trophic structure of animal population. Muscle is considered to be the most common tissue for SIA, however, lipid content in muscle causes a considerable bias to the interpretation of isotopic ratios of animals. Neon flying squid (Ommastrephes bartramii) is an important economic cephalopod of Chinese distant water fishery, and plays a major role in marine ecosystems. In this study, the effects of lipid extraction on stable isotope ratios of the muscles of 53 neon flying squids were investigated and the interference mechanism of lipid in SIA was clarified with the aim of contrasting the suitability of different lipid correction models of stable carbon isotope. Results showed that the stable carbon and nitrogen isotopic values of non-lipid extracted samples significantly increased after lipid extractions by 0.71‰ and 0.47‰, respectively, which suggested that lipid extraction in cephalopod isotope study is needed prior to stable carbon isotope analysis but not recommended for stable nitrogen isotope analysis. The results could help remove the effects of lipid contents and standardize SIA muscle samples, thereby getting better understanding of the isotopic change of neon flying squids in the future.