Captive breeding is one of the most effective ways of ensuring the conservation of several endangered species. However, only a few studies have explored the effects of living in captivity on animals at the molecular level. In the present study, the isobaric tags for relative and absolute quantification (iTRAQ) approach was applied to compare protein expression in the blood of endangered spotted seal pups (Phoca largha) inhabiting different environments. We identified 519 proteins from 2,628 peptides. The expression of 158 proteins significantly differed between seals in temporary captivity and those in the wild. In addition, 140 proteins were differentially expressed between seals in temporary captivity and those in long-term captivity, and 235 proteins were differentially expressed between the wild and long-term captive groups (p < 0.05). In seal pups in the wild, proteins associated with maintaining cell stability and integrity of cardiomyocytes, protecting the heart, and preventing anaemia were upregulated, reflecting the high stress and energetic costs of moving and foraging. In addition, significant differences were detected in immune-related proteins among the three groups. The results of this study expand our understanding of protein expression profiles in spotted seals and provide data to support future conservation of this species.