We have previously demonstrated that the release of some of the 14-3-3 isoforms from keratinocytes is able to influence the expression of key matrix metalloproteinases (MMPs) in dermal fibroblasts. Conversely, in this study we aimed to investigate whether dermal fibroblasts possess the ability to modulate the expression of 14-3-3 proteins in keratinocytes. In order to address this question, human keratinocytes and dermal fibroblasts were harvested and co-cultured. Intra- and extracellular levels of 14-3-3 proteins (β, η, γ, and σ) were analyzed using western blot analysis, and the gene expression was further assessed by quantitative real-time polymerase chain reaction. Gene analysis revealed an up-regulation of all four 14-3-3 isoforms of interest. In addition, the findings of this study reveal a significant increase in the intracellular levels of 14-3-3 γ and σ in keratinocytes co-cultured with fibroblasts compared to those of the mono-cultured control keratinocytes. Mechanistic investigations also demonstrated the capacity of several mitogen-activated protein kinase-specific inhibitors to markedly reduce induction of 14-3-3 σ in keratinocytes stimulated with fibroblast-conditioned medium. The study concluded that dermal fibroblasts possess the ability to influence the expression of several 14-3-3 isoforms (notably γ and σ) in keratinocytes, suggesting that the two cell types might be capable of bi-directionally influencing the protein expression of one another in vivo.