Background: Female neonates are well-recognised to have improved outcomes compared to males. The mechanisms remain poorly understood, but hormonal influences on immune function may contribute to the female advantage during infection and inflammation. The aim of this study was to investigate the in vitro treatment effects of 17β-estradiol (E2) and its antagonism in males versus females on CD14+ monocyte cell surface receptor (TLR4, CD11b, ER-α, ER-β) expression and reactive oxygen species (ROS) production from mononuclear cells in umbilical cord blood (UCB) and adult blood. Methods: UCB samples were collected from term neonates and whole blood was collected from adults (M:F n=10 in each group). Mononuclear cells were isolated via density gradient centrifugation and flow cytometry was used to assess receptor expression and ROS production in the presence of E2, ICI 182,780 (ER antagonist), or lipopolysaccharide (LPS/endotoxin) in various combinations. Results: Basal expression of TLR4, CD11b, ER-α and ER-β did not differ on monocytes between sexes or between adults versus neonates. Treatment with E2, ICI 182,780, or LPS individually or in combination did not modulate CD11b or TLR4 expression in neonates or adults. Higher expression of monocyte ER-β expression was noted in female versus male adults following ICI 182,780 treatment alone (p<0.05). Female neonates exhibited less ROS production following LPS and E2 treatment in combination compared to male neonates (p<0.05). Conclusion: The influence of E2 on neonatal mononuclear cell ROS production provides preliminary evidence for sex-specific disparities in neonatal immune function. These responses may be amenable to immunomodulation.