Extinction learning is modulated by N-methyl d-aspartate receptors (NMDAR) particularly in prefrontal and hippocampal brain regions. The use of of NMDA agonists in exposure therapy of anxiety disorders has been investigated in various patient groups. Behavioral results showed beneficial effects of pre-learning administration of the partial NMDAR agonist d-Cycloserine (DCS) on therapy success. However, the impact of DCS upon non-fear-related contextual extinction, and associated recruitment of extinction-relevant brain regions is as yet unknown. In the present fMRI study, healthy human participants performed a context-related associative learning and extinction task. A single dose of DCS, administered prior to extinction learning, enhanced extinction learning performance in an identical context, and increased activation in prefrontal, temporal as well as hippocampal/insular regions, compared to placebo controls. In contrast, DCS did not affect extinction learning in a novel context, nor the renewal effect, which describes the recovery of an extinguished response if the context of extinction differs from the context of recall. Our findings demonstrate a specific involvement of prefrontal and hippocampal NMDAR in the modification of established stimulus-outcome associations in identical contexts and thus their role in behavioral flexibility, underlining their potential for enhancing AAA extinction learning.