Salidroside, a compound of natural origin, has displayed a broad spectrum of pharmacological properties. This study aimed to evaluate the inhibitory effects of salidroside on glutamate-induced cell death in a primary culture of rat hippocampal neurons as compared to brain-derived neurotrophic factor (BDNF), a usual positive control. MTT and LDH assays, together with Hoechst 33342 staining, terminal deoxynucleotidyl transferase dUTP-mediated nicked end labeling (TUNEL) assay and flow cytometric analysis using annexin-V and propidium (PI) label, indicated that salidroside pretreatment attenuated glutamate-induced apoptotic cell death in primary cultured hippocampal neurons, showing a dose-dependent pattern. Furthermore, caspase-3 activity assay and calcium measurements with Fluo 4-AM, respectively, revealed that salidroside pretreatment antagonized activation of caspase-3 and elevation of intracellular calcium level, both of which were induced by glutamate stimulation, thus adding to the understanding of how salidroside offered neuroprotection against glutamate excitotoxicity.