Renal chloride metabolism is currently poorly understood but may serve as both a diagnostic and a treatment approach for acute kidney injury. We investigated whether plasma chloride, ammonia and glutamine as well as urinary chloride, ammonium and glutamine concentrations may serve as markers for acute kidney injury in paediatric patients. We conducted a prospective observational trial in a tertiary care paediatric intensive care unit. Ninety-one patients after cardiopulmonary bypass surgery were enrolled. Plasma glutamine, creatinine, (serum) albumin, urinary electrolytes and glutamine were collected pre-cardiopulmonary bypass surgery, at paediatric intensive care unit admission, and at 6, 12, 24, 48 and 72 h after paediatric intensive care unit admission. The urinary strong ion difference was calculated. The median urinary chloride excretion decreased from 51 mmol/L pre-cardiopulmonary bypass to 25 mmol/L at paediatric intensive care unit admission, and increased from 24 h onwards. Patients with acute kidney injury had lower urinary chloride excretion than those without. The median urinary strong ion difference was 59 mmol/L pre-cardiopulmonary bypass, rose to 131 mmol/L at 24 h and fell to 20 mmol/L at 72 h. The plasma chloride rose from 105 mmol/L pre-cardiopulmonary bypass to a maximum of 109 mmol/L at 24 h. At 24 h the plasma chloride concentration was associated with the presence of acute kidney injury. There was no association between plasma or urinary amino acids and chloride excretion or kidney injury. In conclusion, renal chloride excretion decreased in all patients, although this decrease was more pronounced in patients with acute kidney injury. Our findings may reflect a response of the kidneys to critical illness, and acute kidney injury may make these changes more pronounced. Targeting chloride metabolism may offer treatment approaches to acute kidney injury.