MMP-9, a member of the matrix metalloproteinase family that degrades collagen IV and processes chemokines and cytokines, participates in epidermal remodeling in response to stress and injury. Limited activity of MMP-9 is essential while excessive activity is deleterious to the healing process. Tumor necrosis factor (TNFα), a key mediator of cutaneous inflammation, is a powerful inducer of MMP-9. Calcitriol, the hormonally active vitamin D metabolite, and its analogs are known to attenuate epidermal inflammation. We aimed to examine the modulation of MMP-9 by calcitriol in TNFα-treated keratinocytes. The immortalized HaCaT keratinocytes were treated with TNFα in the absence of exogenous growth factors or active ingredients. MMP-9 production was quantified by gelatin zymography and real-time RT-PCR. Activation of signaling cascades was assessed by western blot analysis and DNA-binding activity of transcription factors was determined by EMSA. Exposure to TNF a markedly increased the protein and mRNA levels of MMP-9, while pretreatment with calcitriol dose dependently reduced this effect. Employing specific inhibitors we established that the induction of MMP-9 by TNFα was dependent on the activity of the epidermal growth factor receptor, c-Jun-N-terminal kinase (JNK), NFκB and extracellular signal-regulated kinase-1/ 2. The effect of calcitriol was associated with inhibition of JNK activation and reduction of DNA-binding activities of the transcription factors activator protein-1 (AP-1) and NFκB following treatment with TNFα. By down-regulating MMP-9 levels active vitamin D derivatives may attenuate deleterious effects due to excessive TNFα-induced proteolytic activity associated with cutaneous inflammation.