Recent evidence indicates that the NO-related species, nitroxyl anion (NO⁻), is produced in physiological systems by several redox metal–containing proteins, including hemoglobin, nitric oxide synthase (NOS), superoxide dismutase, and S-nitrosothiols (SNOs), which have recently been identified in brain. However, the chemical biology of NO⁻ remains largely unknown. Here, we show that NO⁻—unlike NO^·, but reminiscent of NO⁺ transfer (or S-nitrosylation)—reacts mainly with Cys-399 in the NR2A subunit of the N-methyl-D-aspartate (NMDA) receptor to curtail excessive Ca²⁺ influx and thus provide neuroprotection from excitotoxic insults. This effect of NO⁻ closely resembles that of NOS, which also downregulates NMDA receptor activity under similar conditions in culture.