Skin repair requires the activation of keratinocytes and is mediated by controlled inflammation and cell migration and proliferation, ending with the regeneration of well-differentiated cell layers. Whey derivatives contain galactooligosaccharides (GOS), which have potential beneficial effects on wound healing due to their activity as toll-like receptor ligands, although their direct nonprebiotic effects in the skin have not yet been described. In this study, we investigated the effects of different whey-derived products and purified GOS on a human keratinocyte cell line. We found that the inflammatory cytokine interleukin-8 (IL-8) was upregulated by nuclear factor kappa B (NF-kB) signaling triggered by whey derivatives and GOS and that wound healing was accelerated by promoting cell migration and the loss of E-cadherin in the absence of epithelial–mesenchymal transition. Interestingly, the treatments enhanced the mitochondrial function in association with the translocation of the Forkhead Box O1 (FOXO-1) transcription factor. Finally, we detected the increased expression of the differentiation markers induced by GOS and whey derivatives. All together, our results show that GOS-containing products can promote wound closure and skin health by direct activity on keratinocyte functions. Among the preparations tested, the fermented compound produced by autochthonous microorganisms was the most active in modulating keratinocyte activity, supporting the biological value of whey derivatives for health.