The Na⁺/Ca²⁺ exchanger (NCX) is involved in Ca²⁺ transport across the plasma membrane in excitable cells, exchanging one Ca²⁺ for three Na⁺ depending on the electrochemical gradients. The NCX2 isoform is expressed in the adult brain and is thought to contribute to Ca²⁺ extrusion following neuronal excitation. Enhancing Ca²⁺_i signalling via inhibition of NCX2 has been suggested as a possible therapeutic strategy for cognitive disorders. This study was undertaken to elucidate the contribution of NCX2 to Ca²⁺ clearance, its role in hippocampal function and the possible therapeutic window between plasticity and excitotoxicity. A lentiviral vector expressing a short hairpin RNA targeting rat NCX2 (shNCX2) was generated, validated for NCX2 knockdown efficiency and used to transduce rat primary hippocampal neurons. Transduction of shNCX2 abolished Na⁺– and Ca²⁺-evoked responses in Fura-2 fluorescence imaging experiments, confirming ablation of NCX function. Furthermore, the Ca²⁺_i response to glutamate application was prolonged in these neurons but was unaffected in viral-transduced controls, demonstrating the role of NCX2 in Ca²⁺ clearance following postsynaptic excitation. To investigate if NCX2 knockdown affects hippocampal function, the lentiviral vector was stereotaxically injected into the CA1 region of the hippocampus of adult Lister-Hooded rats (under isoflurane anaesthesia by inhalation). Eight weeks post-injection, ex vivo electrophysiological recordings of field EPSPs in the CA3-CA1 pathway were performed in hippocampal slices. An increase in the paired-pulse ratio was observed in shNCX2-transduced vs. EGFP-transduced slices, providing evidence for enhancement of short-term presynaptic plasticity. Increased [Ca²⁺]_i is a known trigger for excitotoxicity and we therefore tested the effect of NCX2 knockdown on cell viability using the MTT assay, a measure of mitochondrial activity. Reduced cell viability was observed in shNCX2-transduced primary hippocampal neurons the same multiplicity of infection at which enhancement of glutamate-evoked responses was observed. Taken together, these data show that inhibition of NCX2 function enhanced both pre- and postsynaptic Ca²⁺ signalling but concomitantly reduced cell viability.