p16 is an inhibitor of cyclin-dependent kinases and regulating senescence-mediated arrest as well as p21. The expression of p16 has been evaluated in human kidney diseases. However, the regulation of p16 nuclear translocation has yet to be fully investigated. TGF-β1 is well-known to be one of the major cytokines in developing kidney diseases. TGF-β1 can upregulate p21 expression and be involved in the process of senescence. In contrast, the relationship between TGF-β1 and p16 has been poorly investigated. Here, we report the role of TGF-β1-Smad3 pathway to regulate the p16 behavior in glomerular endothelial cells. To clarify the role of TGF-β1 in the regulation of p16, we analyzed podocyte-specific TGF-β1 overexpression mice. In glomeruli, p16 was found in the nuclei of glomerular endothelial cells, leading to endothelial cellular senescence. However, the expression level of p16 was not increased in glomeruli. In cultured endothelial cells, TGF-β1 induced nuclear translocation of p16 without the increase in p16 expression. Among human glomerular diseases, p16 was detected in the nuclei of endothelial cells. In summary, we could show the novel role of podocyte TGF-β1 in the management of p16 behavior and cellular senescence in glomeruli, which has clinical relevance for human glomerular diseases.