Affinity-purified Shiga toxin (Stx) 2 given intraperitoneally to mice caused weight loss and hind-limb paralysis followed by death. Globotriaosylceramide (Gb(3)), the receptor for Stx2, was localized to neurons of the central nervous system (CNS) of normal mice. Gb3 was not found in astrocytes or endothelial cells of the CNS. In human cadaver CNS, we found Gb(3) in neurons and endothelial cells. Mouse Gb(3) localization was confirmed by immunoelectron microscopy. In Stx2-exposed mice, anti-Stx2-gold immunoreaction was positive in neurons. During paralysis, after Stx2 injection, multiple glial nuclei were observed surrounding motoneurons by electron microscopy. Also revealed was a lamellipodia-like process physically inhibiting the synaptic connection of motoneurons. Ca2+ imaging of cerebral astrocytic end-feet in Stx2-treated mouse brains suggested that the toxin increased neurotransmitter release from neurons. In this article, we propose that the neuron is a primary target of Stx2, affecting neuronal function and leading to paralysis.