Spermidine, a natural polyamine presented widely in mammalian cells, has been implicated to extend the lifespan of several model organisms by inducing autophagy. However, the effect of spermidine against neuronal damage has not yet been fully determined. In this study, neuronal cell injury was induced by treating PC12 cells and cortical neurons with 1 μM staurosporine (STS). We found that STS-induced cell injury could be efficiently attenuated by pretreatment with 1 mM spermidine. Spermidine inhibited the caspase 3 activation induced by STS. Moreover, STS incubation resulted in autophagic degradation failure, which could be attenuated by the pretreatment of spermidine. Knocking down the expression of Beclin 1 efficiently suppressed autophagosome and autolysosome accumulation, and abolished the protective effects of spermidine against STS-induced neurotoxicity. Increased Beclin 1 cleavage and partial nuclear translocation of Beclin 1 fragment was detected in STS-treated cells, which could be blocked by spermidine, pan-caspase inhibitor or caspase 3-specific inhibitor. The nuclear translocation of Beclin 1 fragment universally occurs in damaged neurons. Beclin 1 mutation at the sites of 146 and 149 prevented the intracellular re-distribution of Beclin 1 induced by STS. In addition, intraperitoneal injection of spermidine ameliorated ischemia/reperfusion-induced neuronal injury in the hippocampus and cortex of rats, possibly via blocking caspase 3 activation and consequent Beclin 1 cleavage. Our findings suggest that caspase 3-mediated Beclin 1 cleavage occurs in acute neuronal cell injury both in vitro and in vivo. The neuroprotective effect of spermidine may be related to inhibition of the caspase 3-mediated Beclin 1 cleavage and restoration of the Beclin 1-dependent autophagy.