Matrix metalloproteinase-9 (MMP-9) appears to be a major protease responsible for the degradation of matrix and growth-promoting agents in chronic wounds. Honey has been successfully used for treating non-healing wounds associated with infections. However, the mechanisms of its action at the cellular level have remained poorly understood. The aim of this study was to investigate the effect of fir honeydew honey on TNF-α-induced MMP-9 expression and secretion from human keratinocytes (HaCaT) and to identify the honey component(s) responsible for a discovered effect. A C18 solid-phase column was used for preparation of honey aqueous extract (HAE). Expression and production of MMP-9 by HaCaT cells were determined by reverse transcription-PCR, gelatine zymography and Western blot analysis using a polyclonal antibody against MMP-9. We found that HAE inhibited TNF-α-induced production of MMP-9 in keratinocytes in a dose-dependent manner at both the mRNA and protein levels. Apigenin and kaempferol, identified flavonoids in HAE, markedly inhibited MMP-9 production from HaCaT and epidermal keratinocytes. Taken together, fir honeydew honey, which contains certain flavonoids, prevents TNF-α-induced proteolytic activity in cutaneous inflammation. Thus, our findings provide clear evidence that honey may serve as a natural treatment for dermatological problems associated with a persistent inflammation.