We recently showed that 17β-estradiol (E(2)) treatment ameliorated type 2 diabetic glomerulosclerosis in mice in part by protecting podocyte structure and function. Progressive podocyte damage is characterized by foot process effacement, vacuolization, detachment of podocytes from the glomerular basement membrane, and apoptosis. In addition, podocytes are highly dependent on the preservation of their actin cytoskeleton to ensure proper function and survival. Because E(2) administration prevented podocyte damage in our study on diabetic db/db mice and has been shown to regulate both actin cytoskeleton and apoptosis in other cell types and tissues, we investigated whether actin remodeling and apoptosis were prevented in podocytes isolated from E(2)-treated diabetic db/db mice. We performed G-actin/F-actin assays, Western analysis for Hsp25 expression, Ras-related C(3) botulinum toxin substrate 1 (Rac1) activity, and apoptosis assays on previously characterized podocytes isolated from both in vivo-treated placebo and E(2) female db/db mice. We found that in vivo E(2) protects against a phenotype change in the cultured podocytes characterized by a percent increase of F-actin vs. G-actin, suppression of Hsp25 expression and transcriptional activation, increase of Rac1 activity, and decreased apoptotic intermediates. We conclude from these studies that E(2) treatment protects against podocyte damage and may prevent/reduce diabetes-induced kidney disease.