Pregnancy (including puerperium) is a period of hypercoagulability and seems to be an independent major risk factor for venous thromboembolism (VTE). However, the basis of the increased risk of VTE in pregnancy and around delivery is unknown. We hypothesized that changes in PRL, which is a prominently increased hormone during pregnancy and lactation, might be involved in the activation of platelets. To investigate platelet functional abnormalities in pregnancy, we assessed the ADP-stimulated and nonstimulated P-selectin expression of platelets in 42 consecutive pregnant women, 22 normo- and hyperprolactinemic patients with pituitary tumors, and controls. In addition, the aggregation of platelets by human PRL in vitro was studied. We found a significant correlation between PRL values and ADP stimulation of platelets in pregnant women (r = 0.56; P < 0.0001) and patients with pituitary tumors (r = 0.57; P = 0.006). Hyperprolactinemic pregnant women or hyperprolactinemic patients with pituitary tumors revealed significantly higher ADP stimulation of platelets (P < 0.0001) than healthy controls or normoprolactinemic patients with pituitary tumors. These results were reconciled by increased in vitro stimulation and aggregation of platelets using human PRL. Our novel findings demonstrate that hyperprolactinemia causes increased platelet aggregation via ADP stimulation both in vitro and in vivo. Moreover, our data indicate that PRL may be a physiological cofactor of the delicate coagulation balance during pregnancy and puerperium that might explain the increased risk of VTE in pregnant women around delivery. Further studies of the interaction between PRL and platelets will clarify the clinical relevance of hyperprolactinemia as a potential risk factor for VTE.