The difference of metabolite profiles between raw and cooked pufferfish (Takifugu flavidus) meat was explored by H NMR technique and multivariate statistical methods. The orthogonal projection to latent structure-discriminant analysis (OPLS-DA) results showed an obvious separation between two samples. There were 24 dominating metabolites in the pufferfish muscle extraction, of which 11 metabolites changed significantly (p < 0.05), including 9 amino acids (alanine, leucine, methionine, tyrosine, glutamate, glycine, arginine, lysine, taurine), 1 organic acid (lactate) and 1 alkaloid (betaine). Moreover, the Student's t-test was performed to identify the different levels of metabolites. Sensory intensity experiments showed that there was a significant difference in the umami taste between raw and cooked pufferfish meat (p < 0.05). The content of glutamate, aspartate and 5'-IMP in the cooked meat increased, making the umami taste more intense. This study provided essential information about the metabolites explaining the taste difference between raw and cooked pufferfish meat.