<p>Steroid sulfatase (STS) is a key enzyme for the desulfation of steroid sulfates, converting them into their biologically active forms. Notably, X-linked ichthyosis (XLI), a genetic disorder characterized by hyperkeratinization, arises as a direct result of STS deficiency. Keratinocyte differentiation is essential for proper keratinization. In this study, gene ontology analysis from STS-deficient mice revealed enhanced differentiation and upregulation of calcium-related signaling. Calcium plays a key role in regulating keratinocyte differentiation, with STS-deficient cells showing a marked increase in intracellular calcium influx. Additionally, these cells significantly upregulated calcium-sensing receptors (CasR), leading to elevated tyrosine phosphorylation, increased differentiation signaling, and the upregulation of early differentiation markers, including keratin 1 and keratin 10, as seen in HaCaT cells and mouse primary keratinocytes. Furthermore, STS inhibitors enhanced the expression of E-cadherin and terminal differentiation markers such as involucrin and loricrin. Due to increased calcium sensitivity, STS-deficient cells treated with calcium exhibited a significant upregulation of differentiation markers and reduced sensitivity to calcium chelation. Collectively, our findings demonstrate that reduced STS expression and inhibition of its activity enhance calcium responsiveness, induce CasR expression, and amplify calcium signaling, thereby promoting keratinocyte differentiation. These findings offer valuable insights into the mechanisms underlying STS deficiency-induced hyperkeratinization.</p>