Psoriasis is a skin disease characterized by the presence of red plaques on the skin. This pathology is well-known to be a retinoid-sensitive disease. Previous investigations have shown that retinoids can modulate epidermal proliferation with an antiproliferative potential in hyperproliferative skins. The aim of this study was to compare the development of psoriatic substitutes cultured in a retinoic acid supplemented medium with those cultured in medium receiving no supplement, to define the effects of this growth factor on keratinocyte proliferation and differentiation. The self-assembly method was used to create substitutes. Characterization of the psoriatic substitutes was performed by histological and immunolabeling analyses. Results showed that psoriatic keratinocyte substitutes cultured with retinoic acid have a thinner epidermis compared with psoriatic keratinocyte substitutes cultured without this supplement. Further, the expression of all tested cell differentiation markers was restored in psoriatic keratinocyte substitutes cultured in presence of retinoic acid. No significant change in epidermal thickness or in the expression of late differentiation markers was observed in healthy keratinocyte substitutes cultured with or without retinoic acid; however, some changes were reported for proliferation and early differentiation markers. Results suggest that retinoic acid can modulate epidermal differentiation and proliferation with an antiproliferative potential in psoriatic substitutes such as observed in psoriatic skin in vivo.