Energy metabolism and physical performance are dependent on sufficient iron metabolism. Therefore, studies evaluating the iron profile are necessary to elucidate this trace mineral requirements and its role in the equine exercise physiology. The aim of this study was to evaluate the influence of barrel racing exercise on serum iron profile of Quarter horses. Twenty-two regularly trained Quarter horses (8 females and 14 males, 4.8 ± 2.4 years old, mean body weight 431.7 ± 33.9 kg) were enrolled in this study with the owner’s informed consent. All horses were evaluated at T0 (at rest), T1 (immediately after barrel racing trial), T2 (30 min after trial) and T3 (2 h after trial). At these time points, blood samples were taken to determine the packed cell volume (PCV), red blood cell count (RBC), haemoglobin concentration (Hb), total protein (TP), serum iron (SI), total iron-binding capacity (TIBC) and transferrin saturation (TSAT). Variables were analyzed for normality through Kolmogorov-Smirnov test and comparisons were made using Tukey test, considering P < 0.05. The imposed exercise challenge significantly altered PCV, RBC, haemoglobin concentration, TP, SI and TSAT, with higher values were recorded at T1. These findings can be linked to an increased demand as a result of physical activity. Significant changes occurred in the iron status in physically well-conditioned Quarter horses during the barrel racing exercise. Better understanding of iron metabolism in horses will help determine the actual necessity of supplementation.