To investigate the effect of acute hyperglycemia on brain function in adolescents with type 1 diabetes (T1D). Twenty participants with T1D (aged 14.64 ± 1.78 years) and 20 age-matched healthy control subjects (aged 14.40 ± 2.82 years) performed two functional MRI sessions. Participants with T1D performed the first scanning session under euglycemic and the second under hyperglycemic clamp (20 mmol/L 360 mg/dL). Lower spatial working memory (sWM) capacity during acute hyperglycemia and significant differences in activation of regions of interest during different stages of the sWM task ( = 0.014) were observed. Acute hyperglycemia negatively affected sWM capacity in adolescents with T1D, which is relevant for daily functioning and academic performance.