<p>Glucocorticoids are commonly prescribed in clinical settings; however, their prolonged use at high doses can adversely affect human health. One significant complication following glucocorticoid therapy is glucocorticoid-induced osteoporosis (GIO), which is second in incidence only to senile osteoporosis. Based on previous research indicating that Klotho alleviates dexamethasone-induced osteoblast cytotoxicity through the NF-kB pathway, we aimed to explore the underlying mechanisms in greater depth. We assessed the impact of Lithium chloride (LiCl), a Wnt pathway activator, on glucocorticoid-induced cell cytotoxicity and viability. Cytotoxicity was specifically quantified by Annexin V/PI flow cytometry. We performed qRT-PCR and Western blotting analyzes to scrutinize the expressions of genes and proteins associated with both canonical and non-canonical Wnt signaling pathways. Dexamethasone treatment induced an upregulation of the non-canonical Wnt ligand, Wnt5a, and a downregulation of the canonical ligand, Wnt3a, along with its downstream marker, β-catenin. Transfection with Klotho counteracted these effects. Klotho has the potential to modulate both canonical and non-canonical Wnt signaling pathways, thereby counteracting osteoblast cytotoxicity induced by glucocorticoids.</p> Graphical Abstract <p></p>