Podocyte injury is a common cause of massive proteinuria. Astragaloside IV (AS-IV) has been reported to protect podocytes in diabetic models. However, the effects and potential mechanism of AS-IV on puromycin aminonucleoside (PAN)-induced podocyte injury remains unclear. The aim of the present study was to investigate the protective effect of AS-IV on PAN-induced podocyte injury both in vivo and in vitro. In vivo, we induced a podocytic-injury model in rats via a single tail vein injection of PAN. The rats in the treatment group received AS-IV intragastrically (i.g.) at a dose of 40 mg/kg/day for 10 days. At the end of the experiment, 24 h urine, serum and kidney samples were collected for examination. In vitro, we injured podocytes with 30 μg/ml PAN and treated them with AS-IV at concentrations of 5, 25 and 50 μg/ml. Next, we analyzed podocyte protein expression and the Wnt/planar-cell polarity (PCP) pathway using western blot and immunofluorescence (IF). Our results showed that AS-IV decreased proteinuria in PAN-injured rats, and restored specific protein expression in podocytes. In PAN-induced injuries to human podocytes, AS-IV restored the expression and distribution of F-actin and synaptopodin, and repaired the morphology of the actin-based cytoskeleton. Notably, AS-IV could activate the Wnt/PCP pathway by promoting expression of Wnt5a, protein tyrosine kinase 7 (PTK7), Rho-associated coiled-coil-containing protein kinase 1 (ROCK1), Ras-related C3 botulinum toxin substrate 1 (Rac1) and phospho-SAPK/JNK (Thr183/Tyr185) (p-JNK) in vivo and in vitro. In conclusion, we demonstrated that AS-IV alleviated PAN-induced injury to the podocyte cytoskeleton, partially by activating the Wnt/PCP pathway. AJTR Copyright © 2020.