Background: Cardiorespiratory fitness (CRF) and physical activity (PA) are inversely related to the occurrence of type 2 diabetes (T2D). Both play an important role in reducing serum ferritin (SF) concentration. Increased SF concentration is considered a contributing factor for developing T2D. Methods: The present cohort study investigated 5,512 adult participants enrolled in the Aerobics Center Longitudinal Study (ACLS) between 1995 and 2001. The subjects completed a comprehensive medical examination and a SF evaluation, and had been followed up until either diabetes onset, death, or the cut-off date of November 2007. Three CRF levels were categorized. SF quartile levels were defined by gender and menopausal status. The incidence of T2D was calculated for 10,000 person-years, and hazard ratios (HR) were computed to predict the incidence of T2D based on SF quartiles and CRF levels. Results: SF concentration was significantly higher in males than in females (148.5 ± 104.7 ng/ml vs. 52.2 ± 45.9 ng/ml) and was inversely associated with CRF levels. In the high CRF group, 32.7% of participants had a low SF concentration whereas only 16.8% of participants had a high SF concentration level. After adjusting for potential confounders, male participants in the highest SF quartile level had a 1.7 times (HR: 1.67, 95% CI: 1.05, 2.66; p-trend = 0.027) increased risk for developing T2D compared with those in the lowest SF quartile group. Conclusion: Lower SF concentration was associated with lower risk of developing T2D in those regularly participating in CRF. The findings from this study suggest that SF concentration could be used as a diabetic predictor. Based on these results clinicians and public health professionals should promote regular physical activity or fitness to reduce the incidence of T2D.