Vanadate is the hallmark inhibitor of the P-type ATPase family; however, structural details of its inhibitory mechanism have remained unresolved. We have determined the crystal structure of sarcoplasmic reticulum Ca2+-ATPase with bound vanadate in the absence of Ca2+. Vanadate is bound at the catalytic site as a planar VO3− in complex with water and Mg2+ in a dephosphorylation transition-state-like conformation. Validating bound VO3− by anomalous difference Fourier maps using long-wavelength data we also identify a hitherto undescribed Cl− site near the dephosphorylation site. Crystallization was facilitated by trinitrophenyl (TNP)-derivatized nucleotides that bind with the TNP moiety occupying the binding pocket that normally accommodates the adenine of ATP, rationalizing their remarkably high affinity for E2P-like conformations of the Ca2+-ATPase. A comparison of the configurations of bound nucleotide analogs in the E2·VO3− structure with that in E2·BeF3− (E2P ground state analog) reveals multiple binding modes to the Ca2+-ATPase.