Reference values of fecal calprotectin (fCP) have not been convincingly established in children. We aimed to investigate fCP concentrations in a larger population of healthy children aged 4-16 years to analyze more in depth the behavior of fCP in this age range and to determine if cut-off levels could be conclusively recommended. A prospective study was conducted to investigate fCP concentrations of healthy children aged 4-16 years. In 212 healthy children, the median and 95th percentile for fCP were 18.8 mg/kg and 104.5 mg/kg, respectively. We found a statistically significant association between the 95th percentile of fCP concentrations and age (p < 0.001). We propose a nomogram to facilitate the interpretation of fCP results in children aged 4-16 years. Further studies are required to validate the proposed values in clinical practice.