Purpose: This study aimed to determine the acute and chronic effects of interval aerobic exercise on hepcidin, ferritin, and liver enzymes in adolescents with beta-thalassemia major. Methods: Twenty-six beta-thalassemia major adolescents referred to the Thalassemia Clinic and Research Center were selected as study participants and randomly divided into control (n = 13) and training (n = 13) groups. Participants performed 3 sessions per week for 45 minutes in each session for 8 weeks of aerobic interval exercise with an intensity of 50% to 65% of the heart rate reserve. Blood samples were taken before, immediately after the exercise session, and 48 hours after the last training session, and liver enzymes aspartate aminotransferase, alanine aminotransferase (ALT), alkaline phosphatase (ALP), ferritin, and hepcidin were evaluated. Results: The results showed a decrease in aspartate aminotransferase, ALT, ALP, ferritin, and hepcidin levels due to 8 weeks of aerobic interval training (P = .14, P = .97, P = .03, P < .001, P < .001; respectively). Intergroup changes in all variables except ALT and hepcidin were significant (P < .05). Besides, acute aerobic exercise increased levels of aspartate aminotransferase, ALT, ferritin, and hepcidin (P = .04, P = .52, P < .001, P < .001; respectively), whereas ALP levels decreased (P < .001). In addition, changes in ALP and hepcidin levels were significant between the 2 groups (P = .05, P < .001; respectively). Conclusion: Based on the study’s results, it can be concluded that 8 weeks of aerobic interval training can decrease ferritin and hepcidin levels, but acute aerobic exercise increases them.