Collectively, our study demonstrated that overexpression of Sp1 alleviated TGF-β1-induced fibrosis in HK-2 cells by inducing Klotho expression and Sp1 directly modulated Klotho expression in kidney cells by binding to a specific CG-rich site between nt − 430 and − 380 in the KL promoter region, which help to further understand the transcriptional regulation of Klotho in renal disease models. The gene discussed is TGFB1; the disease is kidney disorder.