In microglia, Toll-like receptor 4 (TLR4) is well known to contribute to neuroinflammatory responses following brain ischemia. TLR4 is also expressed in neurons and can mediate the conduction of calcium (Ca2+) influx, but the mechanistic link between neuronal TLR4 signaling and brain ischemic injury is still poorly understood. Here, primary neuronal cell cultures from TLR4 knockout mice and mice with conditional TLR4 knockout in glutamatergic neurons (TLR4cKO) were used to establish ischemic models in vitro and in vivo, respectively. We found that deleting TLR4 would reduce the neuronal death and intracellular Ca2+ increasement induced by oxygen and glucose deprivation (OGD) or lipopolysaccharide treatment. Infarct volume and functional deficits were also alleviated in TLR4cKO mice following cerebral ischemia/reperfusion (I/R). Furthermore, TLR4 and N-methyl-D-aspartate receptor subunit 2B (NMDAR2B) were colocalized in neurons. Deletion of TLR4 in neurons rescued the upregulation of phosphorylated NMDAR2B induced by ischemia via Src kinase in vitro and in vivo. Downstream of NMDAR2B signaling, the interaction of neuronal nitric oxide synthase (nNOS) with postsynaptic density protein-95 (PSD-95) was also disrupted in TLR4cKO mice following cerebral I/R. Taken together, our results demonstrate a novel molecular neuronal pathway in which TLR4 signaling in neurons plays a crucial role in neuronal death and provide a new target for neuroprotection after ischemic stroke.