OBJECTIVE: Klotho and Fibroblast Growth Factor (FGF)-23 were recently postulated as candidate biomarkers and/or therapeutic targets in acute kidney injury (AKI). We examined whether urine Klotho and serum intact FGF23 levels were differentially and independently associated with major adverse kidney events (MAKE) in critically ill patients with and without AKI. DESIGN: Single-center, prospective, case-control study. SETTING: ICU in a tertiary medical center. PATIENTS: 54 AKI patients and 52 controls without AKI admitted to the ICU. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: AKI was defined by KDIGO criteria and included only AKI stage ≥2. Controls were matched by age, gender, and baseline eGFR. Paired serum and urine samples were obtained 24-48h after AKI diagnosis (cases) or ICU admission (controls). The primary outcome was 90-day MAKE, which was the composite of all-cause death, dependence on renal replacement therapy or a 50% or higher decrease in eGFR from baseline. Forty-four (41.5%) patients developed MAKE-90. Patients who developed MAKE-90 had more comorbidity, higher acuity of illness scores and more prevalent AKI. Levels of urine Klotho adjusted by creatinine (Cr) were lower and serum intact FGF23 levels were higher in AKI patients vs. ICU controls. In adjusted models, the highest vs. lowest tertile of urine Klotho/Cr was independently associated with an overall 95% lower risk of MAKE-90 (81% lower risk in patients with AKI). The highest vs. lowest tertile of serum intact FGF23 was associated with >300% higher risk of MAKE-90. CONCLUSIONS: Urine Klotho/Cr levels were significantly lower and serum intact FGF23 levels significantly higher in critically ill patients with AKI vs. matched-controls without AKI. When measured in the first 48h of ICU admission or AKI diagnosis, urine Klotho/Cr independently associated with major adverse kidney events, particularly in patients with AKI. These results show promise for testing these biomarkers -individually or in combination- as part of novel risk-prediction models of renal outcomes in the ICU.