Protein hydrolysate from frame, bone and skin (FBSH) of threadfin bream was prepared using Virgibacillus sp. SK33 proteinase and fractionated using sequential ultrafiltration membranes with molecular weight cut-offs (MWCO) of 30, 5 and 1kDa, respectively. Four fractions, namely FBSH-I (>30kDa), FBSH-II (5–30kDa), FBSH-III (1–5kDa), and FBSH-IV (<1kDa), were obtained. All fractions were rich in Lys, Glu/Gln, Gly, Pro, Ala, Asp/Asn, and Arg. FBSH-III and FBSH-IV showed the highest surface hydrophobicity measured by 8-anilino-1-naphthalenesulfonic acid (ANS) probe (p<0.05). FBSH-III showed the highest antioxidant activity and cytoprotective effects against tert-butyl hydroperoxide (TBHP)-induced cytotoxicity of Caco-2 cells. In addition, FBSH-III inhibited lactate dehydrogenase (LDH) leakage and intracellular reactive species (ROS) production in a dose-dependent manner. FBSH-III retained antioxidant activity and cytoprotective capacity after in vitro simulated gastrointestinal digestion. These results suggested that FBSH-III might potentially be nutraceutical peptides with antioxidative properties.