Keratinocytes, suffer numerous biochemical reactions during the transition from basal layer to stratum corneum (SC), including synthesis of specific basal (K14) and suprabasal (K10) keratins and cornified envelope-associated proteins. Epidermal differentiation complex (EDC) is constituted through cross-linking of involucrin, loricrin, profilaggrin, among others, on intracellular surface of plasma membrane in upper spinous and granular layers of epidermis1-2. Studies has shown many factors, such skin inflammatory process and prolonged exposure to sunlight, could affect the skin barrier and extracellular matrix inducing a decrease in the synthesis of the major dermal proteins, collagen and elastin, clinically characterized by wrinkles, rough skin and loss of water and skin tone. In this study, we evaluated the effects of a moisturizing formulation on the production of envelope proteins filaggrin, involucrin, loricrin, keratin 10 and 14, and extracellular matrix collagen and elastin using an in vitro model of human keratinocytes and fibroblasts. Moisturizing formulation system with a unique, three dimensional glycol-matrix delivery system that releases moisturizing molecules sequentially into the stratum corneum, resulting in a highly significant improvement of both immediate and long term skin hydration containing Glycerin; Enteromorpha Compressa Extract; Hydrolyzed Caesalpinia Spinosa Gum; Caesalpinia Spinosa Gum; Rheum Rhaponticum Root Extract and Tocophery Acetate, was provided by 1MANTECORP SKIN CARE, CECI – Innovation and Consumer Studies Center, Barueri/SP – Brazil. Human dermal fibroblasts and keratinocytes were incubated for 48 hours with three concentrations of the moisturizing formulation previously determined by XTT method. The levels of collagen and elastin were measured respectively in fibroblast culture supernatant and cell lysate using a precipitation commercial kit (Biocolor, UK). The synthesis of filaggrin, involucrin loricrin, keratin 10 and keratin 14 was measured in supernatant from keratinocyte culture using a colorimetric competitive enzyme-linked immunosorbent assay – ELISA (Uscn Life Science Inc., Houston, Tx, USA). The one-way analysis of variance (ANOVA) test was used followed by Bonferroni post-test with a 5% significance level. Our results demonstrated that incubation of fibroblasts cultures with the moisturizing formulation increased the synthesis of extracellular matrix proteins collagen and elastin up to 43.33% and 41.62%, respectively, in relation to non-treated group. The incubation of keratinocytes cultures with the moisturizing formulation increased levels of involucrin, filaggrin, loricrin, keratin 10 and keratin 14, reaching-up to 43.84%, 37.04%, 32.02%, 20.89% and 18.05%, respectively, in relation to non-treated control. The water content of the SC and skin surface lipids is important factors in the appearance and function of skin barrier3. Traditional moisturizing ingredients are known to decelerate the loss of skin humidity, increase hydration of the SC and improve physical and chemical properties of skin surface, making it moist, smooth and soft4. However, most of the products claimed for skin moisturizing produce an immediate and transient effect. Thus, optimized skin care cosmetics should be developed to supply a proper moisturizing effect and the same time being capable to balance the synthesis of skin barrier proteins5 and to protect against down-regulation of extracellular matrix. In this study, we evaluated the ability of moisturizing formulation in the production of proteins involved in stratum corneum and extracellular matrix integrity. Moisturizing formulation increased the levels of all proteins evaluated. Our results indicate a biological activity of this skin care product in the promotion of cellular hydration and skin barrier integrity.