This study aimed to investigate how running water (rapid, R) and ice-water (slow, I) thawing methods affect the quality of Takifugu rubripes fillets. Thawing shrinkage and changes in extractable proteins quantified by tandem mass tag (TMT)-labeled quantitative proteomics were compared. The results showed that the rapidly thawed fillets were quickly underwent greater shrinkage, and the smaller gap areas were reduced by 7.5 % compared to slow thawing. Compared with fresh fish fillets, the outflow of proteins such as ATP synthase, NADH dehydrogenase, and aconitase within mitochondria increased in both thawing methods that presents cell membrane damage and significant disruptions in mitochondrial structure. The pyruvate dehydrogenase and cytochrome c were significantly upregulated in slow-thawing group. Whereas myosin and structural proteins including the Z-line related were significantly upregulated in the rapid-thawing group. These differential proteins serve as crucial markers for elucidating mechanism involved in muscle quality deterioration under different thawing conditions.