The underlying mechanism of the role of mitochondria in color changing of tilapia fillet during 0-4 d storage is not completely clear. A total of 209 differentially significant expressed proteins (DSEPs) were identified by using label-free mitochondrial proteomics, with 56 proteins up-regulated in T2 and 61 proteins (up-regulated) in T3. Protein-Protein interaction reveled proteins which participate in TCA cycles (Citrate synthase (cs)), Oxidoreductase (Malate dehydrogenase (mdh1, mdh2), Succinyl-CoA (Oxct1), Hydroxyacyl-coenzyme a dehydrogenase (hadh), Dehydrogenase/reductase (SDR family) member 1 (dhrs1)) interacted strongly with each other. In turn, they can increase the level of mitochondrial respiration and mitochondrial function, leading to color changing of tilapia fillet. The heat shock 60kD protein 1 (chaperonin, hspd1) interacted with metabolic enzymes (cs and mdh2) and had important effects on color. These results could help researchers better understand the color changing mechanism on the surface of tilapia fillet during the storage.