Vascular endothelial growth factor C (VEGF-C) is a critical activator of tumor lymphangiogenesis that recently has been strongly implicated in the tumor metastasis process. In this study, we identified that HRG-β1 stimulated up-regulation of VEGF-C mRNA and protein of human breast cancer cells in a dosage- and time-dependent manner and that this up-regulation was de novo RNA synthesis-dependent. The HRG-β1-induced increase in VEGF-C expression was effectively reduced by treatment with Herceptin, an antibody specifically against HER2. Also, when HER2 was overexpressed in MCF-7 cells that resulted in an evident increase in the VEGF-C level, suggesting an essential role of HER2 in mediating VEGF-C up-regulation by HRG-β1. NF-κB has been shown to be probably involved in interleukin-1β- or tumor necrosis factor-α-induced VEGF-C mRNA expression in human fibroblasts. Here we found that HRG-β1 could stimulate NF-κB nuclear translocation and DNA-binding activity via the IκBα phosphorylation-degradation mechanism. Blockage of the NF-κB activation cascade caused a complete inhibition of the HRG-β1-induced elevation of VEGF-C. In promoter-reporter assay, the luciferase activities of the reporter constructs, including the putative NF-κB site deleted and mutated form were significantly reduced after HRG-β1 treatment as compared with the 1.5-kb VEGF-C promoter. Although investigating the upstream kinase pathway(s) involved in HRG-β1-elicited NF-κB activation and VEGF-C up-regulation, we found that HRG-β1 could activate extracellular signal-regulated protein kinase 1/2, phosphatidylinositol 3′-kinase, and p38 mitogen-activated protein kinase (MAPK) in MCF-7. However, only SB203580 (a specific inhibitor of p38 MAPK), not PD98059 nor LY294002, blocked the up-regulation of VEGF-C by HRG-β1. A similar inhibition in VEGF-C expression was obtained by cell transfection with dominant-negative p38 (p38AF). Interestingly, the HRG-β1-induced NF-κB activation cascade was also effectively blocked by SB203580 treatment or p38AF transfection. Our data thus suggests that HRG-β1 stimulated a NF-κB-dependent up-regulation of VEGF-C through the p38 MAPK signaling pathway in human breast cancer cells.