Abstract Background Purinergic P2Y₁ and P2Y₁₂ receptors (P2Y₁-R and P2Y₁₂-R) are G protein-coupled receptors (GPCR) activated by adenosine diphosphate (ADP) to mediate platelet activation, thereby playing a pivotal role in hemostasis and thrombosis. While P2Y₁₂-R is the major target of antiplatelet drugs, no P2Y₁-R antagonist has yet been developed for clinical use. However, accumulating data suggest that P2Y₁-R inhibition would ensure efficient platelet inhibition with minimal effects on bleeding. In this context, an accurate characterization of P2Y₁-R antagonists constitutes an important preliminary step. Results Here, we investigated the pharmacology of P2Y₁-R signaling through Gq and β-arrestin pathways in HEK293T cells and in mouse and human platelets using highly sensitive resonance energy transfer-based technologies (BRET/HTRF). We demonstrated that at basal state, in the absence of agonist ligand, P2Y₁-R activates Gq protein signaling in HEK293T cells and in mouse and human platelets, indicating that P2Y₁-R is constitutively active in physiological conditions. We showed that P2Y₁-R also promotes constitutive recruitment of β-arrestin 2 in HEK293T cells. Moreover, the P2Y₁-R antagonists MRS2179, MRS2279 and MRS2500 abolished the receptor dependent-constitutive activation, thus behaving as inverse agonists. Conclusions This study sheds new light on P2Y₁-R pharmacology, highlighting for the first time the existence of a constitutively active P2Y₁-R population in human platelets. Given the recent interest of P2Y₁₂-R constitutive activity in patients with diabetes, this study suggests that modification of constitutive P2Y₁-R signaling might be involved in pathological conditions, including bleeding syndrome or high susceptibility to thrombotic risk. Thus, targeting platelet P2Y₁-R constitutive activation might be a promising and powerful strategy for future antiplatelet therapy.