<p>Zn²⁺ is essential for neuronal signaling, but imbalance cause cell death and neurodegenerative disorders. While the buffering system maintains low cytosolic Zn²⁺ concentration ([Zn²⁺]_i), the details on physiological stimuli elevating [Zn²⁺]_i for neuronal processes remain limited. Our previous reports have demonstrated that dopamine elevates [Zn²⁺]_i through the cAMP−NO pathway, activating autophagy and inflammation in neurons. In this study, we adopted the Zn²⁺ imaging technique to verify how glutamate elevated [Zn²⁺]_i in cultured cortical neurons and examined the inflammatory response. Our results showed that glutamate elevates the [Zn²⁺]_i, by activating ionotropic glutamate receptors. Inhibitors of calmodulin (CaM), CaM-dependent protein kinase II (CaMKII), and NO synthase (NOS) blocked the glutamate-induced Zn²⁺ response. High-K⁺ buffer induced-membrane depolarization significantly elevated the intracellular Ca²⁺ concentration ([Ca²⁺]_i) but only slightly increased [Zn²⁺]_i and NO production. Glutamate also transiently increased NOS phosphorylation at Ser¹⁴¹⁷ within 15&#xa0;min. The Zn²⁺ chelator, TPEN suppressed glutamate-induced inflammasome formation. These results indicate that glutamate-induced local increment in [Ca²⁺]_i via the ionotropic glutamate receptors activates the CaM−CaMKII−NOS complex to produce NO and elevate [Zn²⁺]_i. which trigger inflammation in cultured neurons. Henceforth, this novel glutamate−Zn²⁺ signaling pathway after glutamate depolarization elevates [Ca²⁺]_i indicates the involvement of Zn²⁺ in modulating long-term neuronal activities.</p>