Mucosal healing is an important treatment goal in Crohn's disease. We investigated the association between serum infliximab trough levels and mucosal healing, and the infliximab cut-off levels required for mucosal healing in paediatric patients. In this multicentre, retrospective, cross-sectional study, medical records and electronic data of paediatric patients with luminal Crohn's disease, who had received infliximab for ≥1 year, were examined. Ileocolonoscopy was performed on the same day as the infliximab infusion, and serum samples for trough levels were collected immediately before infusion. Mucosal healing was defined as a Simple Endoscopic Score for Crohn's Disease of 0. Univariate, multivariate logistic regression, and receiver operating characteristic curve analyses were performed. Overall, 105 patients [median age 14.8 years] were included, with mucosal healing observed in 48.6%. Median serum infliximab trough levels were higher in patients with mucosal healing [4.5 µg/mL] than without [3.3 µg/mL, p = 0.002]. In the final multivariate model, infliximab trough level ≥4.2 µg/mL [p = 0.002] and ≥1-year duration from diagnosis to infliximab treatment [p = 0.003] were positively and negatively associated with mucosal healing, respectively. The infliximab trough level for achieving mucosal healing with a specificity of 80% was ≥5 µg/mL. Associations between serum infliximab trough concentrations and mucosal healing were observed in paediatric patients. Identification of the infliximab trough level that positively associates with mucosal healing in most paediatric patients with Crohn's disease [≥5 µg/mL] may guide treatment decisions to optimise therapeutic response in the era of treat-to-target.