Iron deficiency anemia (IDA) is a nutritional deficiency disease with a high incidence rate worldwide. Bioactive peptides are safe and effective, have multiple functions and can serve as potential candidates for alleviating IDA. In this study, the anti-anemia effects of tuna dark muscle peptides were explored in a diet-induced IDA mouse model. The results showed that tuna dark muscle peptides alleviated the IDA phenotype, oxidative stress and iron metabolism. In addition, tuna dark muscle peptides reversed gut microbiota dysbiosis in IDA mice. Furthermore, the transplanted fecal microbiota from tuna dark muscle peptide-treated mice also alleviated IDA symptoms and regulated iron metabolism and the gut microbiota, indicating that the anti-anemic effects were at least partially mediated by the gut microbiota. Thus, we identified a new and safe prebiotic material to alleviate IDA and provided ideas for the development of peptides. At the same time, these data also provided a theoretical basis for fecal microbiota transplantation to alleviate IDA.