Cholesterol has been recently suggested to regulate the early steps of keratinocyte differentiation through lipid rafts. In many cell types, depletion of cholesterol activates signaling proteins like epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), or extracellular signal-regulated kinase (ERK) known to affect cell differentiation. In this study, we explored the effects of cholesterol depletion on the phenotype of cultured keratinocytes, using a treatment with methyl-β-cyclodextrin (MβCD) to extract cholesterol and a treatment with lovastatin to inhibit cholesterol neosynthesis. Analysis of the expression of differentiation marker genes in early differentiating confluent cultures reveals that cholesterol depletion induces downregulation of keratin 14 (K14) and keratin 10 (K10) and upregulation of involucrin. MβCD treatment induces phosphorylation of EGFR, HER2, and ERK, but not KER3. Inhibition of EGFR with PD153035 impairs the MβCD-induced phosphorylation of EGFR, HER2, and ERK, but does not impair the alteration of K14, K10, or involucrin gene expression, indicating that other signaling proteins regulate this phenomenon. p38 has been suggested to regulate the expression of involucrin during keratinocyte differentiation. We found that MβCD treatment induces a prolonged phosphorylation of p38 in general and p38α in particular. An inhibition of p38 with PD169316 impairs the upregulation of involucrin mRNAs by a treatment with MβCD, but not by a p38δ-activating TPA treatment, which might suggest that cholesterol depletion alters involucrin gene expression through activation of p38α/β.