Objective— Estrogens exert beneficial effect on the blood vascular system. However, their role on the lymphatic system has been poorly investigated. We studied the protective effect of the 17β estradiol—the most potent endogenous estrogen—in lymphedema—a lymphatic dysfunction, which results in a massive fluid and fat accumulation in the limb. Approach and Results— Screening of DNA motifs able to mobilize ERs (estrogen receptors) and quantitative real-time polymerase chain reaction analysis revealed that estradiol promotes transcriptional activation of lymphangiogenesis-related gene expression including VEGF (vascular endothelial growth factor)-D, VEGFR (VEGF receptor)-3, lyve-1, and HASs (hyaluronan synthases). Using an original model of secondary lymphedema, we observed a protective effect of estradiol on lymphedema by reducing dermal backflow—a representative feature of the pathology. Blocking ERα by tamoxifen—the selective estrogen modulator—led to a remodeling of the lymphatic network associated with a strong lymphatic leakage. Moreover, the protection of lymphedema by estradiol treatment was abrogated by the endothelial deletion of the receptor ERα in Tie2-Cre; ERα ^lox/lox mice, which exhibit dilated lymphatic vessels. This remodeling correlated with a decrease in lymphangiogenic gene expression. In vitro, blocking ERα by tamoxifen in lymphatic endothelial cells decreased cell–cell junctions, inhibited migration and sprouting, and resulted in an inhibition of Erk but not of Akt phosphorylation. Conclusions— Estradiol protection from developing lymphedema is mediated by an activation of its receptor ERα and is antagonized by tamoxifen. These findings reveal a new facet of the estrogen influence in the management of the lymphatic system and provide more evidence that secondary lymphedema is worsened by hormone therapy.