Background: Cardiopulmonary bypass (CPB) may lead to adrenal insufficiency (AI). Emerging evidence supports association of AI with morbidity after cardiac surgery. Aims: The aim of this study was to define AI incidence in neonates undergoing complex cardiac surgery with CPB and its association with intraoperative post-CPB outcomes. Methods: Forty subjects enrolled in a prior randomized control trial who received preoperative methylprednisolone as part of our institutional neonatal bypass protocol were included. No intraoperative steroids were given. ACTH stimulation tests were performed: preoperatively and 1 h after separation from CPB. AI was defined as <9 μg·ml−1 increase in cortisol at 30 min post cosyntropin 1 mcg. Clinical outcomes were collected up to 90 min after CPB. Results: 2/40 (5%) subjects had preoperative AI vs 13/40 (32.5%) post-CPB AI, P ≤ 0.001. No significant difference was observed in age, gestational age, weight, CPB time, circulatory arrest, or STAT category between subjects with or without post-CPB AI. ACTH decreased from preoperative values 127.3 vs 35 pcg·ml−1 [median difference = 81.8, 95% CI = 22.7–127.3], while cortisol increased from 18.9 vs 75 μg·dl−1 [median difference = 52.2, 95% CI = 36.3–70.9]. Post-CPB AI was associated with increased median colloid resuscitation, 275 vs 119 ml·kg−1 [median difference = 97.8, 95% CI = 7.1–202.2]; higher median peak lactate, 9.4 vs 6.9 mg·dl−1 [median difference = 3.2, 95% CI = 0.04–6.7]; median post-CPB lactate, 7.9 vs 4.3 mg·dl−1, [median difference 3.6, 95% CI = 2.1–4.7], and median lactate on admission to CICU, 9.4 vs 6.0 mg·dl−1 [median difference = 3, 95% CI = 1.1–4.9]. No difference was observed in blood pressure or vasoactive inotrope score at any time point measured in operating room (OR). Higher initial post-CPB cortisol correlated with decreased cosyntropin response. Conclusions: Neonatal cardiac surgery with CPB and preoperative methylprednisolone leads to AI as determined by low-dose ACTH stimulation test in one-third of patients. AI is associated with increased serum lactate and colloid resuscitation in OR. Impact of preoperative methylprednisolone on results is not defined. Benefit of postoperative steroid administration in neonates with post-CPB AI warrants further investigation.