During thrombopoiesis, megakaroycytes undergo extensive cytoskeletal remodeling to form proplatelet extensions that eventually produce mature platelets. Proplatelet formation is a tightly orchestrated process that depends on dynamic regulation of both tubulin reorganization and Rho-associated, coiled-coil containing protein kinase/RhoA activity. A disruption in tubulin dynamics or RhoA activity impairs proplatelet formation and alters platelet morphology. We previously observed that protein kinase Cepsilon (PKCϵ), a member of the protein kinase C family of serine/threonine-kinases, expression varies during human megakaryocyte differentiation and modulates megakaryocyte maturation and platelet release. Here we used an in vitro model of murine platelet production to investigate a potential role for PKCϵ in proplatelet formation. By immunofluorescence we observed that PKCϵ colocalizes with a/b-tubulin in specific areas of the marginal tubular-coil in proplatelets. Moreover, we found that PKCϵ expression escalates during megakarocyte differentiation and remains elevated in proplatelets, whereas the active form of RhoA is substantially downregulated in proplatelets. PKCϵ inhibition resulted in lower proplatelet numbers and larger diameter platelets in culture as well as persistent RhoA activation. Finally, we demonstrate that pharmacological inhibition of RhoA is capable of reversing the proplatelet defects mediated by PKCϵ inhibition. Collectively, these data indicate that by regulating RhoA activity, PKCϵ is a critical mediator of mouse proplatelet formation in vitro.