Myofibrillar protein accounting for about 60% of total muscle proteins is expected to be a promising source of bioactive peptides. The purpose of the present study was to purify antioxidant peptides from myofibrillar protein hydrolysate of chicken breast by ultrafiltration and gel filtration chromatography, and evaluate their chemical antioxidant activities and protective effects in H2O2-stressed NIH-3T3 cells. Four major peptides were identified using nano-LC-ESI-MS/MS as ITTNPYDY, IGWSPLGSL, ITTNPYDYHY, and LRVAPEEHPTL. The sequenced peptides were synthesized and exhibited remarkable radical-scavenging ability, ORAC (108.2-133.5 μM TE per mg peptide), and FRAP (75.4-92.5 mM Fe2+ per mg peptide). Structure-activity relationship indicated that the antioxidant capacity of the peptides was more related to the presence of hydrophobic and antioxidant amino acids (including Trp, Val, Ile, Leu, Ala, Pro, Gly, Asp, His, and Tyr) in the sequences as well as their molecular structures. Moreover, they protected NIH-3T3 cells against oxidative damage through inhibiting ROS generation and lipid peroxidation. Especially, the antioxidant peptides ITTNPYDY and IGWSPLGSL significantly (p < 0.05) elevated intracellular glutathione level and antioxidant enzyme activities, and suppressed apoptosis by blocking caspase-3 activation. This work highlights that the selected peptides may serve as functional food ingredients with antioxidant and cytoprotective characteristics.