Rett syndrome (RS), a progressive severe neurodevelopmental disorder mainly caused by de novo mutations in the X-chromosomal MeCP2 gene encoding the transcriptional regulator methyl-CpG-binding protein 2, is a leading cause of mental retardation with autistic features in females. However, its pathogenesis remains incompletely understood, and no effective therapy is available to date. We hypothesized that a systemic oxidative stress may play a key role in the pathogenesis of classic RS. Patients with classic RS (n = 59) and control subjects (n = 43) were evaluated. Oxidative stress markers included intraerythrocyte non-protein-bound iron (NPBI; i.e., free iron), plasma NPBI, F2-isoprostanes (F2-IsoPs, as free, esterified, and total forms), and protein carbonyls. Lung ventilation/perfusion (V/Q) ratio was assessed using a portable gas analyzer, and RS clinical severity was evaluated using standard scales. Significantly increased intraerythrocyte NPBI (2.73-fold), plasma NPBI (× 6.0), free F2-IsoP (× 1.85), esterified F2-IsoP (× 1.69), total F2-IsoP (× 1.66), and protein carbonyl (× 4.76) concentrations were evident in RS subjects and associated with reduced (− 10.53%) arterial oxygen levels compared to controls. Biochemical evidence of oxidative stress was related to clinical phenotype severity and lower peripheral and arterial oxygen levels. Pulmonary V/Q mismatch was found in the majority of the RS population. These data identify hypoxia-induced oxidative stress as a key factor in the pathogenesis of classic RS and suggest new therapeutic approaches based on oxidative stress modulation.