Ciclopirox olamine (CPX), an off-patent antifungal agent used to treat mycoses of skin and nails, has recently been demonstrated to be a potential anticancer agent. However, the underlying mechanism is not well understood. Here for the first time we show that CPX inhibited lymphangiogenesis in an in vitro model (tube formation). This effect was in part associated with inhibition of vascular endothelial growth factor receptor 3 (VEGFR-3) expression, as overexpression of VEGFR-3 conferred partial resistance to CPX inhibitory effect on tube formation in lymphatic endothelial cells (LECs), whereas downregulation of VEGFR-3 mimicked the effect of CPX, blocking the tube formation. Further study revealed that CPX did not alter mRNA level, but inhibited protein synthesis and promoted protein degradation of VEGFR-3. In addition, we found that CPX inhibited phosphorylation of the extracellular signal-related kinase 1/2 (ERK1/2), a downstream effector of VEGFR-3. Overexpression of VEGFR-3 attenuated CPX inhibition of ERK1/2 phosphorylation, whereas downregulation of VEGFR-3 inhibited extracellular signal-related kinase 1/2 (ERK1/2) phosphorylation in LECs. Ectopic expression of constitutively active mitogen -activated protein kinase kinase 1 (MKK1) resulted in activation of ERK1/2, and partially prevented CPX inhibition of LEC tube formation. The results suggest that CPX inhibits LEC tube formation at least in part through inhibiting VEGFR-3-mediated ERK signaling pathway.