Background: The mechanisms underlying neonatal platelets hyporesponsiveness are not fully understood. While previous studies have demonstrated developmental impairment of agonist-induced platelet activation, differences in inhibitory signaling pathways have been scarcely investigated. Objective: To compare neonatal and adult platelets with regard to inhibition of platelet reactivity by prostaglandin E1 (PGE<sub>1</sub>). Methods: Platelet-rich plasma from umbilical cord (CB) or adult blood was incubated with PGE<sub>1</sub> (0–1 μM). We assessed aggregation in response to adenosine diphosphate (ADP), collagen, and thrombin receptor activating peptide as well as cyclic adenosine 3′5′-monophosphate (cAMP) levels (ELISA). G<sub>αs</sub>, G<sub>αi2</sub>, and total- and phospho-protein kinase A (PKA) were evaluated in adult and CB ultrapure and washed platelets, respectively, by immunoblotting. Results: Neonatal (vs. adult) platelets display hypersensitivity to inhibition by PGE<sub>1</sub> of platelet aggregation induced by ADP and collagen (PGE<sub>1</sub> IC<sub>50</sub>: 14 and 117 nM for ADP and collagen, respectively, vs. 149 and 491 nM in adults). They also show increased basal and PGE<sub>1</sub>-induced cAMP levels. Mechanistically, PGE<sub>1</sub> acts by binding to the prostanoid receptor IP (prostacyclin receptor), which couples to the G<sub>αs</sub> protein-adenylate cyclase axis and increases intracellular levels of cAMP. cAMP activates PKA, which phosphorylates different target inhibitor proteins. Neonatal platelets showed higher basal and PGE<sub>1</sub>-induced cAMP levels, higher G<sub>αs</sub> protein expression, and a trend to increased PKA-dependent protein phosphorylation compared to adult platelets. Conclusion: Neonatal platelets have a functionally increased PGE<sub>1</sub>-cAMP-PKA axis. This finding supports a downregulation of inhibitory when going from neonate to adult contributing to neonatal platelet hyporesponsiveness.