Summary Background In addition to their key role in hemostasis, platelets and megakaryocytes regulate immune and inflammatory responses, in part through their expression of Toll‐like receptors (TLRs). Among the TLRs, TLR3 recognizes dsRNA associated with viral infection. Thrombocytopenia is a frequent complication of viral infection. However, the expression and functionality of TLR3 in megakaryocytes and platelets is not yet well understood. Objective To study the expression and functionality of TLR3 in the megakaryocytic lineage. Methods and results RT‐PCR, flow cytometric and immunofluorescence assays showed that TLR3 is expressed in CD34+ cells, megakaryocytes, and platelets. Immunoblotting assays showed that stimulation of megakaryocytes with two synthetic agonists of TLR3, Poly(I:C) and Poly(A:U), activated the nuclear factor‐κB (NF‐κB), phosphoinositide 3‐kinase (PI3K)/Akt, extracellular signal‐related kinase (ERK)1/2 and p38 pathways. TLR3–megakaryocyte activation resulted in reduced platelet production in vitro and interferon‐β release through the PI3K–Akt and NF‐κB signaling pathways. TLR3 ligands potentiated the aggregation mediated by classic platelet agonists. This effect was also observed for ATP release, but not for P‐selectin or CD40L membrane exposure, indicating that TLR3 activation was not involved in α‐granule release. In addition, TLR3 agonists induced activation of the NF‐κB, PI3K–Akt and ERK1/2 pathways in platelets. Reductions in platelet production and platelet fibrinogen binding mediated by Poly(I:C) or Poly(A:U) were prevented by the presence of an inhibitor of the TLR3–dsRNA complex. Conclusions Our findings indicate that functional TLR3 is expressed in CD34+ cells, megakaryocytes, and platelets, and suggest a potential role for this receptor in the megakaryopoiesis/thrombopoiesis alterations that occur in viral infections.