Eukaryotic assimilatory nitrate reductase (NR) is a dimeric multidomain molybdo-heme-flavo protein that catalyzes the first and rate-limiting step in the nitrate assimilation of plants, algae, and fungi. Nitrate reduction takes place at the N-terminal molybdenum cofactor-containing domain. Reducing equivalents are derived from NADH, which reduce the C-terminal FAD domain followed by single-electron transfer steps via the middle heme domain to the molybdenum center. In plants, nitrate reduction is post-translationally inhibited by phosphorylation and subsequent binding of 14-3-3 protein to a conserved phosphoserine located in the surface-exposed hinge between the catalytic and heme domain. Here we investigated Arabidopsis thaliana NR activity upon phosphorylation and 14-3-3 binding by using a fully defined in vitro system with purified proteins. We demonstrate that among different calcium-dependent protein kinases (CPKs), CPK-17 efficiently phosphorylates Ser534 in NR. Out of eight purified Arabidopsis 14-3-3 proteins, isoforms ω, κ, and λ exhibited the strongest inhibition of NR. The kinetic parameters of noninhibited, phosphorylated NR (pNR) and pNR in a complex with 14-3-3 were investigated. An 18-fold reduction in k(cat) and a decrease in the apparent K(M)(nitrate) (from 280 to 141 μM) were observed upon binding of 14-3-3 to pNR, suggesting a noncompetitive inhibition with a preferential binding to the substrate-bound state of the enzyme. Recording partial activities of NR demonstrated that the transfer of electrons to the heme is not affected by 14-3-3 binding. The Ser534Ala variant of NR was not inhibited by 14-3-3 proteins. We propose that 14-3-3 binding to Ser534 blocks the transfer of electrons from heme to nitrate by arresting the domain movement via hinge 1.