Objective To investigate the protective effect of pertussis toxin (PTx) on neurons after ischemic stroke and possible mechanisms.Methods C57BL6 mice were used to establish a model of stroke by middle cerebral artery occlusion (MCAO) and were then randomly divided into experimental group and control group, with 12 mice in each group. The mice in the experimental group were given intraperitoneal injection of PTx 1000 ng dissolved in 1 ml normal saline after stroke, and those in the control group were given intraperitoneal injection of 1 ml normal saline. TTC staining was performed 24 hours later to measure infarct area, and immunohistochemistry was used to measure neuronal apoptosis. Primary neurons were cultured in vitro and glutamate stimulation was performed to mimic neuronal injury after stroke. MTT assay and lactate dehydrogenase (LDH) release assay were used to observe the influence of PTx on the survival and injury of neurons after glutamate stimulation, and then the influence of PTx on glutamate-induced neuronal calcium influx was examined.Results PTx treatment reduced cerebral infarct area from 51%±11% to 34%±8% in mice after stroke (P<0.05). Immunohistochemistry showed that PTx reduced the number of caspase-3-positive cells from 677.7±117.8 cells/mm2 in the control group to 297.5±83.6 cells/mm2 in the experimental group (P<0.05) and reduced cell apoptosis. In vitro results showed that PTx increased the viability of neurons after glutamate stimulation, and MTT absorbance was increased from 0.618±0.06 to 1.1±0.12 (P<0.05); at the same time, PTx reduced the release of LDH, and the absorbance of LDH was reduced from 1.31±0.11 to 0.76±0.08 (P<0.05). PTx slowed down and reduced calcium influx into neurons, and after PTx treatment, calcium influx was reduced from 5 times to 2-3 times of the baseline value; the time to half-peak concentration was increased from 18.5±2.5 s to 85.4±10.2 s.Conclusions PTx can reduce calcium influx into neurons after stroke and thus alleviate neuronal injury and reduce infarct area.