Genistein (Gen), a derivative of soy isoflavone aglycone, has been shown to exert significant protective effect on Aβ-induced neurotoxicity and neuroinjury. However, its underlying mechanism remains elusive. The objective was to investigate the inhibitory effect of Gen on Aβ-induced neurotoxicity and to elucidate the underlying mechanism. Primary rat hippocampal neurons were pre-treated with Gen for 2 hr followed by incubation with Aβ 25–35 for an additional 24 hr. The cell viability was assessed by MTT assay. The content and activity of α-, β-secretase and protein kinase C (PKC) were measured, and the antagonistic effect of PKC inhibitor Myr was also analysed to clarify the molecular mechanism of Gen inhibition of Aβ-induced toxicity to hippocampal neurons. The results showed that pre-treatment with Gen significantly increased the cell viability and presented the best effect at the final concentration of 0.375 μg/mL. Gen increases the activity of α-secretase but down-regulates the β-secretase activity. It also enhances the expression and activity of PKC. Myr, a PKC inhibitor, partially blocks the activation effect of Gen. Gen exerts protective effect on Aβ-induced neurotoxicity via activating the PKC signalling pathway, which further regulates the activities of α- and β-secretase and thereby inhibits the formation and toxicity of Aβ.