The expression of matrix metalloproteinases (MMPs) has been implicated in the invasion and metastasis of cancer cells. Here we examined the effect of ascochlorin, a prenyl-phenol anti-tumor compound from the fungus Ascochyta viciae, on the regulation of signaling pathways that control MMP-9 expression in human renal carcinoma (Caki-1) cells. Ascochlorin reduced the invasive activity of Caki-1 cells and inhibited phorbol 12-myristate 13-acetate-induced increases in MMP-9 expression and activity in a dose-dependent manner. Reporter gene, electrophoretic mobility shift, kinase inhibitor assays, and in vitro kinase assay showed that ascochlorin inhibits MMP-9 gene expression by suppressing activation of the nuclear transcription factor activator protein-1 (AP-1) via the extracellular signal-regulated kinase 1 and 2 pathway. The AP-1 family member most specifically affected by ascochlorin was Fra-1. Ascochlorin did not affect the activation of the c-Jun N-terminal or p38 kinase pathways. Moreover, transfection of Caki-1 cells with AP-1 decoy oligodeoxynucleotides resulted in the suppression of phorbol 12-myristate 13-acetate-induced MMP-9 expression and invasion. In conclusion, ascochlorin represents a unique natural anti-tumor compound that specifically inhibits MMP-9 activity through suppression of AP-1-dependent induction of MMP-9 gene expression.