Background Plasma clearance of iohexol is a reliable method to assess the glomerular filtration rate (GFR). The relationship between aging and GFR in horses is unclear. Hypothesis/Objectives To compare GFR estimated by iohexol clearance in horses of different ages. Animals Twenty-one clinically healthy horses were enrolled. Methods Prospective study. Groups: (A) composed by 8 young horses (<= 14 years old) with serum creatinine <1.5 mg/dL, (B) by 7 aged horses (>= 20 years old) with serum creatinine <1.5 mg/dL, and (C) by 6 aged horses (>= 20 years old) with serum creatinine >= 1.5 mg/dL. Iohexol was injected (75.5 mg/kg) through an IV catheter, and plasma samples were collected 5, 30, and 90 minutes later. Plasma clearance of iohexol was obtained by the high-performance liquid chromatography-ultraviolet method. Results The GFR was 2., 2.1, and 1.45 mL/min/kg (median value) in Groups A, B, and C, respectively. Statistical analysis showed differences between Group A versus C for urea, Group A versus B and A versus C for creatinine, and A versus C for GFR. Conclusions and Clinical Importance Glomerular filtration rate was significantly reduced in aged horses with serum creatinine >= 1.5 mg/dL compared to young horses with creatinine <1.5 mg/dL; no differences were obtained between young and aged horses with creatinine <1.5 mg/dL. Glomerular filtration rate evaluation should be considered in aged horses even if the plasma creatinine values are normal.