This article focuses on deciphering the effect of myostatin (MSTN) on podocyte apoptosis in membranous nephropathy (MN) and fathoming out its underlying mechanism. Rats received the intravenous injection of cationized-bovine serum albumin to induce MN in vivo, while angiotensin II (Ang II) was exposed to AB8/13 cells to induce MN model in vitro. The mRNA expression of MSTN was detected by qRT-PCR. The effects of MSTN silencing on MN model rats and cells were assessed by cell counting kit-8 assay, flow cytometry, hematoxylin and eosin staining, and TUNEL assay. The expressions of proteins related to apoptosis and Smad3/protein kinase A (PKA)/NADPH oxidase 4 (NOX4) signaling pathway were examined by western blot. As a result, MSTN was highly expressed in MN cell and rat models. Besides, knockdown of MSTN elevated the MN cell viability and dwindled apoptosis rate, as well as attenuated kidney injury in MN rats. Meanwhile, MSTN silencing lessened the expressions of phosphorylated (p)-Smad3 and Nox4, while boosting the p-PKA expression in MN rats and cells. Additionally, Smad3 overexpression reversed the above effects of MSTN silencing on Ang II-induced podocytes. In conclusion, MSTN knockdown restrains the podocyte apoptosis through regulating Smad3/PKA/NOX4 signaling pathway.