Matrine (Mat) and oxymatrine are two major alkaloids of the Chinese herb Sophora flavescens Ait. (Leguminosae). Previous study has demonstrated that Mat reduces brain edema induced by focal cerebral ischemia. More recently, oxymatrine has been reported to produce neuroprotective effects against focal cerebral ischemia via inhibiting the activation of NF-κB in the ischemic brain tissue. In the current study, we investigated whether direct protection on neurons and astrocytes via inhibition of NF-κB signaling pathway is associated with Mat's neuroprotective effects against cerebral ischemia. In a model of permanent middle cerebral artery occlusion (pMCAO), Mat (12.5, 25 and 50 mg/kg) reduced the infarction volume and improved the neurological deficits in a dose-dependent manner, administered 10 min, 3h and even 6h following pMCAO. Mat 50 mg/kg also decreased the hemispheric water content. The number of GFAP-positive cells was markedly decreased in the ischemic cortex at 12h after ischemia. In contrast, Mat increased the number of GFAP-positive cells. Mat 50mg/kg has no effect on the cerebral blood flow (CBF). Primary neuron or astrocyte cultures were exposed to a paradigm of ischemic insult by using an oxygen-glucose deprivation (OGD), Mat (50-200 μM) reduced LDH leakage and the number of neuronal and astrocytic apoptosis, and increased the number of MAP2-positive and GFAP-positive cells. Further observations revealed that Mat increased the protein levels of IκBα, and blocked the translocation of NF-κB p65 from the cytosol to the nucleus in the ischemic cortex and injured neurons and astrocytes induced by in vitro OGD. Moreover, Mat could down-regulate NF-κB p65 downstream pro-apoptotic gene p53 and/or c-Myc in the injured neurons and astrocytes induced by OGD. The present findings suggest that Mat, even when administrated 6h after ischemia, has neuroprotective effects against focal cerebral ischemia and directly protects neurons and astrocytes via inhibition of NF-κB signaling pathway, contributing to matrine's neuroprotection against focal cerebral ischemia.