Chronic UV exposure is responsible for long term clinical manifestations such as photoaging and photocancers. One of the major events involved in the development of skin photodamage is up-regulation of matrix metalloproteinase-1 (MMP-1). In this study, the effects of a water-soluble extract of Rosmarinus officinalis (Ro) on the expression of UV-induced MMP-1 were investigated. Using MMP-1 promoter-reporter gene constructs, Ro extract was shown to inhibit UV-induced up-regulation of MMP-1 gene transcription. The anti-MMP-1 effect was confirmed at the protein level in dermal human fibroblasts exposed either to UVB, UVA or Solar Simulated Radiation. Zymographic analysis on casein and gelatin gels revealed that Ro extract more specifically targeted MMP-1 compared to MMP-2. Using a 3D-skin model exposed to Solar Simulated Radiation, anti-MMP-1 activity was confirmed together with a photoprotective effect at the morphological level. Finally the release of cytokines IL1alpha and IL6 which participate in the up-regulation of MMP-1 induced by UV exposure could be prevented by the Ro extract. All together, from molecular to tissue level, these results illustrate the ability of the Ro water-soluble extract to inhibit UV-induced MMP-1 and its potential benefits in preventing cutaneous photodamage.