OBJECTIVES: Evaluate the independent and synergistic associations of fluid overload and acute kidney injury with outcome in critically ill pediatric patients. DESIGN: Secondary analysis of the Acute Kidney Injury in Children Expected by Renal Angina and Urinary Biomarkers (NCT01735162) prospective observational study. SETTING: Single-center quaternary level PICU. PATIENTS: One-hundred forty-nine children 3 months to 25 years old with predicted PICU length of stay greater than 48 hours, and an indwelling urinary catheter enrolled (September 2012 to March 2014). Acute kidney injury (defined by creatinine or urine output on day 3) and fluid overload (≥ 20% on day 3) were used as outcome variables and risk factors for ICU endpoints assessed at 28 days. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Acute kidney injury and fluid overload occurred in 19.4% and 24.2% respectively. Both acute kidney injury and fluid overload were associated with longer ICU length of stay but neither maintained significance after multivariate regression. Delineation into unique fluid overload/acute kidney injury classifications demonstrated that fluid overload patients experienced a longer ICU and hospital length of stay and higher rate of mortality compared with fluid overload patients, regardless of acute kidney injury status. Fluid overload/acute kidney injury patients had increased odds of death (p = 0.013). After correction for severity of illness, ICU length of stay remained significantly longer in fluid overload/acute kidney injury patients compared with patients without both classifications (17.4; 95% CI, 11.0-23.7 vs 8.8; 95% CI, 7.3-10.9; p = 0.05). Correction of acute kidney injury classification for net fluid balance led to acute kidney injury class switching in 29 patients and strengthened the association with increased mechanical ventilation and ICU length of stay on bivariate analysis, but reduced the increased risk conferred by fluid overload for mortality. CONCLUSIONS: The current study suggests the effects of significant fluid accumulation may be delineable from the effects of acute kidney injury. Concurrent fluid overload and acute kidney injury significantly worsen outcome. Correction of acute kidney injury assessment for net fluid balance may refine diagnosis and unmask acute kidney injury associated with deleterious downstream sequelae. The unique effects of fluid overload and acute kidney injury on outcome in critically ill patients warrant further study.