The RhoA effector mDia1 is involved in controlling the balance between filamentous and monomeric actin, but its role in modulating thrombin-induced actin remodeling and platelet spreading on fibrinogen matrices remains unclear. In this study, mDia1 was shown to translocate to the platelet cytoskeleton following thrombin stimulation, in a phosphoinositide 3-kinase (PI 3-kinase)-dependent manner. Anti-mDia1 loading or pretreatment with PI 3-kinase inhibitors essentially abrogated thrombin-elicited actin stress fiber formation, with a corresponding decrease in the proportion of platelets exhibiting a fully spread morphology. We also investigated the mechanisms underlying the effects of mDia1 on thrombin-induced actin remodeling and platelet spreading, and found that these involved PI 3-kinase-mediated induction of mDia1 interaction with RhoA. Collectively, these results suggest that the PI 3-kinase/RhoA/mDia1 axis is a critical pathway for coupling thrombin signaling to actin cytoskeletal remodeling during platelet spreading.