Traumatic brain injury (TBI) has become a major health concern worldwide, and the poor outcome of TBI increases the need for therapeutic improvement. Secondary injuries following TBI, including excitotoxicity, lead to synaptic dysfunction and provide potential targets for intervention. Postsynaptic scaffold proteins, which are involved in the regulation of excitotoxicity after neuronal injury, play a crucial role in modulating synaptic function. Therefore, exploring the role of postsynaptic scaffold proteins in TBI might uncover new treatments. In this study, we demonstrated that downregulated expression of the postsynaptic scaffold protein Preso protects against neuronal injury after TBI in vitro and in vivo, and these effects are related to the inhibition of N-methyl-D-aspartate receptor (NMDAR) function. Further study showed that Preso facilitates signaling from NMDAR to nitric oxide (NO) and calcium (Ca2+) responses. First, the complex constituting NMDAR, postsynaptic density-95 (PSD-95), and neuronal nitric oxide synthase (nNOS) was shown to be involved in the Preso regulation of the NO response. Uncoupling the linkage between Preso and PSD-95 attenuated the stability of this complex and suppressed the regulatory effect of Preso on the NO response. In addition, phosphorylation of NMDAR by cyclin-dependent kinase 5 (CDK5) was shown to be responsible for the Preso-mediated Ca2+ response, which was dependent on the interaction between Preso and CDK5. These results suggested that the association of Preso with NMDAR signaling can serve as a target for neuroprotection against TBI.