Neuropathic pain represents a significant clinical problem because it is a chronic condition often refractory to available therapy. Therefore, there is still a strong need for new analgesics. Botulinum neurotoxin A (BoNT/A) is used to treat a variety of clinical diseases associated with pain. Glia are in continuous bi-directional communication with neurons to direct the formation and refinement of synaptic connectivity. This review addresses the effects of BoNT/A on the relationship between glia and neurons under neuropathic pain. The inhibitory action of BoNT/A on synaptic vesicle fusion that blocks the release of miscellaneous pain-related neurotransmitters is known. However, increasing evidence suggests that the analgesic effect of BoNT/A is mediated through neurons and glial cells, especially microglia. In vitro studies provide evidence that BoNT/A exerts its anti-inflammatory effect by diminishing NF-κB, p38 and ERK1/2 phosphorylation in microglia and directly interacts with Toll-like receptor 2 (TLR2). Furthermore, BoNT/A appears to have no more than a slight effect on astroglia. The full activation of TLR2 in astroglia appears to require the presence of functional TLR4 in microglia, emphasizing the significant interaction between those cell types. In this review, we discuss whether and how BoNT/A affects the spinal neuron-glia interaction and reduces the development of neuropathy.