Antagonism of N-methyl-D-aspartate glutamatergic receptors (NMDAR) may represent an effective antidepressant mechanism. D-cycloserine (DCS) is a partial agonist at the NMDAR-associated glycine modulatory site that at high doses acts as a functional NMDAR antagonist. Twenty-six treatment-resistant major depressive disorder patients participated in a double blind, placebo-controlled, 6-wk parallel group trial with a gradually titrated high dose (1000 mg/d) of DCS added to their antidepressant medication. DCS treatment was well tolerated, had no psychotomimetic effects and led to improvement in depression symptoms as measured by Hamilton Depression Rating Scale (HAMD; p = 0.005) and Beck Depression Inventory (p = 0.046). Of the 13 subjects treated with DCS, 54% had a ≥ 50% HAMD score reduction vs. 15% of the 13 patients randomized to placebo (p = 0.039). A significant (p = 0.043) treatment× pre-treatment glycine serum levels interaction was registered. These findings indicate that NMDAR glycine site antagonism may be a cost-effective target for development of mechanistically novel antidepressants. Larger-sized DCS trials are warranted.