Accumulation of β-amyloid peptide (Aβ) in the brain plays an important role in the pathogenesis of Alzheimer’s disease (AD). Previous studies have demonstrated the neuroprotective role of osthole against oxygen and glucose deprivation in cortical neurons. However, the effects of osthole on Aβ-induced neurotoxicity in neural cells have rarely been reported. The current study was designed to investigate the protective effects of osthole on a cell model of AD insulted by exogenous Aβ_25-35 and the potential mechanism(s). In this study, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, immunofluorescence analysis, apoptosis assay, reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) techniques were used in primary cortical neurons and SH-SY5Y cells. Our data showed that osthole reduced intracellular Aβ levels in neural cells, which was associated with decreased BACE1 protein; osthole reversed exogenous Aβ_25-35-induced cell viability loss, apoptosis, and synapsin-1 reduction, which was related to the reestablishment of phosphorylation of cyclic AMP response element-binding protein (CREB). The collective evidence indicates that osthole possesses the ability to protect cortical neurons and SH-SY5Y cells against Aβ injury, and the underlying mechanism may be attributed to the enhancement of CREB phosphorylation.