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Acetaminophen is widely used in PICUs. Although randomized controlled trials suggest that acetaminophen significantly reduces body temperature in adults, the effect of acetaminophen on temperature in critically ill children has not been previously quantified.Retrospective observational cohort study.Single-center general and cardiac PICU in a specialist children’s hospital.All children who received acetaminophen or had a fever (temperature ≥ 38°C) while on the ICU over a 40-month period (September 2012 to December 2015).None.In total, 58,177 doses of acetaminophen were administered, with temperature data available for analysis for 54,084 doses. Temperature decreased by 0.11°C (95% CI, 0.09–0.14°C) 4 hours post acetaminophen dose, after adjustment for weight and illness severity. In children who had a fever and were given acetaminophen, temperature decreased by 0.78°C (95% CI, 0.74–0.82°C). Temperature decreased by 0.88°C (95% CI, 0.85–0.92°C) in children who had fever but did not receive acetaminophen. The change in temperature associated with fever was significantly different between those who did and did not receive acetaminophen (likelihood ratio statistic 246.06; p < 2.2 × 10–16).Acetaminophen is associated with a significant decrease in temperature in children with fever. However, temperature may decrease following fever without acetaminophen in the PICU. The threshold to use acetaminophen must be understood to determine the true effect on temperature in any future trials.