Podocyte apoptosis contributes to the pathogenesis of diabetic nephropathy (DN). However, the mechanisms that mediate hyperglycemia-induced podocyte apoptosis remain poorly understood. Recent findings indicate that the disruption of the cytoskeleton is related to the podocyte apoptosis. In the present study, we investigated the involvement of nestin, an important cytoskeleton-associated class VI intermediate filament (IF) protein, in the high glucose (HG)-induced podocyte apoptosis. Our data showed that HG decreased the expression level of nestin, either mRNA or protein, in a time-dependent manner in cultured podocytes. Also, through knockdown of nestin expression by miRNA interference, the HG-induced podocyte apoptotic rate was significantly increased. The expression of cleaved caspase-3 was also markedly elevated. Considering that nestin is a substrate of cyclin-dependent kinase 5 (Cdk5), we further assessed the expression of Cdk5 in HG-treated podocytes. The results showed that HG stimulation increased the protein and mRNA expression of Cdk5 in a time-dependent manner in cultured mouse podocytes. The protein activator of Cdk5, p35, was also increased in a time-dependent manner by HG stimulation, and downregulation of Cdk5 by miRNA interference attenuated the nestin reduction in HG-treated podocytes; the HG-induced podocyte apoptosis, the increased cleaved caspase-3 expression and the Bax/Bcl-2 ratio were all effectively attenuated. These data suggested that nestin, which is dependent on Cdk5 regulation, plays a cytoprotective role in HG-induced podocyte apoptosis.