Objective Neonatal cardiac surgery requiring cardiopulmonary bypass results in a heightened inflammatory response. Perioperative glucocorticoid administration is commonly used in an attempt to reduce the inflammatory cascade, although characterization of the cytokine response to steroids in neonatal cardiac surgery remains elusive because of highly variable approaches in administration. This randomized trial was designed to prospectively evaluate the effect of specific glucocorticoid dosing protocols on inflammatory markers in neonatal cardiac surgery requiring cardiopulmonary bypass. Methods Neonates scheduled for cardiac surgery were randomly assigned to receive either 2-dose (8 hours preoperatively and operatively, n = 36) or single-dose (operatively, n = 32) methylprednisolone at 30 mg/kg per dose in a prospective double-blind trial. The primary outcome was the effect of these steroid regimens on markers of inflammation. Secondary analyses evaluated the association of specific cytokine profiles with postoperative clinical outcomes. Results Patient demographics, perioperative variables, and preoperative indices of inflammation were similar between the single- and 2-dose groups. Preoperative cytokine response after the 2-dose methylprednisolone protocol was consistent with an anti-inflammatory effect, although this did not persist into the postoperative period. Premedication baseline levels of interleukin-6, interleukin-8, interleukin-10, and tumor necrosis factor α were predictive of postoperative intensive care unit and hospital length of stay. Only interleukin-8 demonstrated a postoperative response associated with duration of intensive care unit and hospital stay. Conclusions The addition of a preoperative dose of methylprednisolone to a standard intraoperative methylprednisolone dose does not improve markers of inflammation after neonatal cardiac surgery. The routine administration of preoperative glucocorticoids in neonatal cardiac surgery should be reconsidered.