Amentoflavone is a well-known biflavonoid that has diverse biological effects. Previously, we reported that amentoflavone suppressed UVB-induced matrix metalloproteinase-1 (MMP-1) expression in normal human fibroblasts (NHF). We investigated the effects of amentoflavone on UVB-induced MMP-1 expression in order to elucidate its mode of action. NHF were treated with amentoflavone for indicated times and doses with UVB irradiation. The expressions of MMP-1 gene and protein were determined by RT-PCR and ELISA, respectively. MAP kinase phosphorylation and the expression of c-Fos protein were determined by Western blot. The treatment of amentoflavone completely blocked the upregulation of MMP-1 which is induced by UVB irradiation in HaCaT-NHF co-culture in a dose-dependent manner as well as in NHF monoculture. Also, amentoflavone inhibited UVB-induced activation of extracellular signal-regulated kinase (ERK) without changing total ERK protein level, and did not affect p38 or JNK activation. Finally, AP-1 transcription factor components, phospho-c-Jun and c-Fos protein expressions were decreased by amentoflavone treatment. The major finding of this study shows that amentoflavone inhibits intracellular cell signaling ERK pathway leading to the prevention of MMP-1 expression in human skin fibroblasts. Therefore, these results strongly suggest that amentoflavone should be investigated as a potential agent for the prevention and the treatment of skin photoaging.