Phosphorylation is one of the major mechanisms for posttranscriptional modification of proteins. The addition of a compact, negatively charged moiety to a protein can significantly change its function and localization by affecting its structure and interaction network. We have used all-atom Molecular Dynamics simulations to investigate the structural consequences of phosphorylating the Na(+)/K(+)-ATPase (NKA) residue Ser(936), which is the best characterized phosphorylation site in NKA, targeted in vivo by protein kinase A (PKA). The Molecular Dynamics simulations suggest that Ser(936) phosphorylation opens a C-terminal hydrated pathway leading to Asp(926), a transmembrane residue proposed to form part of the third sodium ion-binding site. Simulations of a S936E mutant form, for which only subtle effects are observed when expressed in Xenopus oocytes and studied with electrophysiology, does not mimic the effects of Ser(936) phosphorylation. The results establish a structural association of Ser(936) with the C terminus of NKA and indicate that phosphorylation of Ser(936) can modulate pumping activity by changing the accessibility to the ion-binding site.