BACKGROUND: Acquired lymphedema is a global health concern with limited treatment options. While vascular endothelial growth factor C (VEGF-C) administration has shown promise for the treatment of this patient population, no small-molecule compounds have hitherto been identified to improve lymphedema by stimulating VEGF-C expression and lymphangiogenesis. OBJECTIVE: This study investigated the therapeutic effect of notoginsenoside R1 (R1) on a mouse model of tail acquired lymphedema and explored the underlying mechanisms. METHODS: C57BL/6J mice and lymphatic endothelial cells (LECs) specific VEGFR-3 knockout transgenic mice underwent surgical induction of tail acquired lymphedema. Tail circumference, lymphatic drainage function, VEGF-C expression, and lymphangiogenesis were measured. LECs' function was assessed using wound healing and tube formation assays. Quantitative PCR (q-PCR) and western blot were conducted to measure VEGF-C expression levels. In addition, RNA sequencing analysis and western blot were performed to elucidate the signal pathways involved. Luciferase reporter assays assessed VEGF-C promoter activity. RESULTS: R1 treatment improved lymphedema, lymphatic function, and lymphangiogenesis in the mouse model. R1 enhanced migration, tube formation, and VEGF-C expression of LECs. These effects were abolished by VEGF-C siRNA and VEGFR-3 inhibitors. VEGFR3 knockout in LECs completely blocked R1's ability to promote lymphangiogenesis and lymphatic drainage while partially but significantly reducing its improvement on lymphedema. R1 activated the cAMP/PKA signaling pathway, leading to PKA and CREB phosphorylation. The PKA inhibitor and CREB siRNA inhibited R1-induced VEGF-C expression. Additionally, R1 activated VEGF-C promoter activity in a CREB-dependent manner. CONCLUSION: R1 emerges as the first reported small natural compound to promote VEGF-C expression. It reduces acquired lymphedema and enhances lymphangiogenesis via the cAMP/PKA/CREB signaling pathway. These findings suggest R1 as a potential novel oral medication for treating acquired lymphedema patients.