The lymphatic vasculature functions to maintain tissue perfusion homeostasis. Defects in its formation or disruption of the vessels result in lymphedema, the effective treatment of which is hampered by limited understanding of factors regulating lymph vessel formation. Mice lacking T1/podoplanin, a lymphatic endothelial cell trans-membrane protein, have malformed lymphatic vasculature with lymphedema at birth, but the molecular mechanism for this phenotype is unknown. Here we show, using primary human lung microvascular lymphatic endothelial cells (HMVEC-LLy), that small interfering RNA (siRNA) mediated silence of podoplanin gene expression has the dramatic effect of blocking capillary tube formation in Matrigel^TM. In addition, localization of phosphorylated ezrin/radixin/moesin (ERM) proteins to plasma membrane extensions, an early event in the capillary morphogenic program in lymphatic endothelial cells, is impaired. We find that cells with decreased podoplanin expression fail to properly activate the small GTPase RhoA early (by 30 minutes) after plating on Matrigel^TM, and Rac1 shows a delay in its activation. Further indication podoplanin action is linked to RhoA activation is that use of a cell permeable inhibitor of RhoA inhibited lymphatic endothelial capillary tube formation in the same manner as did podoplanin gene silencing, which was not mimicked by treatment with a Rac1 inhibitor. These data clearly demonstrate that early activation of RhoA in the lymphangiogenic process, which is required for the successful establishment of the capillary network, is dependent on podoplanin expression. To our knowledge, this is the first time that a mechanism has been suggested to explain the role of podoplanin in lymphangiogenesis.