To investigate the incidence of hemolysis and its association with outcome in neonates with congenital diaphragmatic hernia (CDH) requiring venovenous extracorporeal membrane oxygenation (ECMO) treatment using a Medos Deltastream circuit with a DP3 pump, a hilite 800 LT oxygenator system, and a ¼' tubing. Plasma free hemoglobin (PFH) was prospectively measured once daily during ECMO using spectrophotometric testing. Patients (n=62) were allocated into two groups according to presence or absence of hemolysis. Hemolysis was defined as PFH ≥ 50mg/dL on at least 2 consecutive days during ECMO treatment. Hemolysis was classified as either moderate with a maximum PFH of 50-100mg/dL or severe with a maximum PFH >100mg/dL. Hemolysis was detected in 14 patients (22.6%). Mortality was 100% in neonates with hemolysis compared with 31.1% in neonates without hemolysis (P<.001). In 21.4% hemolysis was moderate and in 78.6% severe. Using multivariable analysis, hemolysis (hazard ratio: 6.8; 95%CI: 1.86-24.86) and suprasystemic pulmonary hypertension (PH) (hazard ratio: 3.07; 95%CI: 1.01-9.32) were independently associated with mortality. Hemolysis occurred significantly more often using 8 French (Fr) cannulae than 13 Fr cannulae (43% vs 17%; P=.039). Cutoff for relative ECMO flow to predict hemolysis were 115ml/kg/ minute for patients with 8 Fr cannulae (Area under the curve [AUC] 0.786, P=.042) and 100ml/kg/ minute for patients with 13 Fr cannulae (AUC 0.840, P<.001). Hemolysis in CDH neonates receiving venovenous ECMO is independently associated with mortality.