The emerging role of TRPC6 as a central contributor to various pathologic processes affecting podocytes has generated interest in the development of therapeutics to modulate its function. Recent insights into the regulation of TRPC6 have revealed protein kinase G (PKG) as a potent negative modulator of TRPC6 conductance and associated signaling via its phosphorylation at 2 highly conserved amino acid residues, threonine 69 (Thr69 in murines; Thr70 in humans) and serine 321 (Ser321 in murines; Ser322 in humans). Here, we tested the role of PKG in modulating TRPC6-dependent responses in primary and conditionally immortalized mouse podocytes. TRPC6 was phosphorylated at Thr69 in non-stimulated podocytes but this declined upon angiotensin II (Ang II) stimulation or overexpression of a constitutively active calcineurin phosphatase (CA-Cn). Ang II induced podocyte motility in an in vitro wound-assay, and this was reduced 30-60% in cells overexpressing a phosphomimetic mutant TRPC6 (TRPC6T70E/S322E) or activated PKG (p<0.05). Pretreatment of podocytes with the PKG agonists SNAP (NO-donor), 8Br-cGMP, Bay 41-2772 (soluble guanylate cyclase activator), or phosphodiesterase 5 inhibitor 4-{[3',4'-(Methylenedioxy)benzyl]amino}[7]-6-methoxyquinazoline (PDE5i), attenuated Ang II-induced Thr69 de-phosphorylation and also inhibited TRPC6-dependent podocyte motility by 30-60%. These data reveal that PKG activation strategies including PDE5 inhibition ameliorate Ang II-induced podocyte dysmotility by targeting TRPC6 in podocytes, highlighting the potential therapeutic utility of these approaches to treat hyperactive TRPC6-dependent glomerular disease.