<b><i>Introduction:</i></b> Prader-Willi syndrome (PWS) is characterized by a switch from failure to thrive to excessive weight gain and hyperphagia in early childhood. An elevated, more unfavorable ratio between acylated and unacylated ghrelin (AG/UAG ratio) might play a role in the underlying mechanisms of this switch. We aimed to assess the evolution of the appetite-regulating hormones acylated ghrelin (AG) and unacylated ghrelin (UAG) and the AG/UAG ratio and their association with the change in eating behavior in children with PWS, compared to healthy age-matched controls. <b><i>Methods:</i></b> A longitudinal study was conducted in 134 children with PWS and 157 healthy controls, from the Netherlands, France, and Belgium. Levels of AG and UAG and the AG/UAG ratio were measured and nutritional phases as reported for PWS were scored. <b><i>Results:</i></b> The AG/UAG ratio was lower in the first years of life in PWS than in controls and started to increase from the age of 3 years, resulting in a high-normal AG/UAG ratio compared to controls. The AG levels remained stable during the different nutritional phases (<i>p</i> = 0.114), while the UAG levels decreased from 290 pg/mL in phase 1a to 137 pg/mL in phase 2b (<i>p</i> &lt; 0.001). The AG/UAG ratio increased significantly from 0.81 in phase 2a to 1.24 in phase 2b (<i>p</i> = 0.012). <b><i>Conclusions:</i></b> The change from failure to thrive to excessive weight gain and hyperphagia in infants and children with PWS coincides with an increase in AG/UAG ratio. The increase in AG/UAG ratio occurred during phase 2a, thus before the onset of hyperphagia.