Matrix metalloproteinase-9 (MMP-9), which degrades the extracellular matrix (ECM), plays an important role in breast cancer pathogenesis. Previously, we reported that the Radix clematidis extract (RCE) inhibits MMP expression by suppressing the nuclear factor-κB (NF-κB) pathway. The purpose of the present study was to investigate the effects of RCE on 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced MMP-9 expression and cell invasion in MCF-7 cells. The toxicity of RCE was determined by MTT assay. MMP-9 expression was determined by real-time PCR, zymography and Western blot analysis. NF-κB activation was assayed by electrophoretic mobility shift assay (EMSA). Results showed that the expression of MMP-9 and cell invasion in response to TPA was increased, whereas TPA-induced MMP-9 expression and cell invasion was decreased by RCE. Moreover, RCE suppressed NF-κB activation in TPA-treated MCF-7 cells. Thus, RCE is a potent inhibitor of TPA-induced MMP-9 expression and markedly blocks the NF-κB pathway in MCF-7 cells.