NG2-glia are an abundant population of cells in the adult CNS that make up a novel glial cell type. Here, we have examined calcium signals in NG2-glia identified by expression of the fluorescent protein DsRed under the control of the NG2 promoter in the white matter of the mouse optic nerve. We focused on mice aged postnatal day (P)12–16, after the main period of oligodendrocyte generation. Using fluo-4 and fura-2 calcium imaging in isolated intact nerves, we show that glutamate and ATP evoke Ca2+ signals in NG2-glia in situ, acting on AMPA-type glutamate receptors and P2Y1 and P2X7 purine receptors; NMDA evoked a weak Ca2+ signal in a small proportion of NG2-glia. We show that axonal action potentials and mechanical stimulation of astrocytes effect the release of glutamate and ATP to act on NG2-glia; ATP alone evokes robust Ca2+ signals, whereas glutamate did not unless AMPA receptor desensitization was blocked with cyclothiazide. We identify the precise contacts that NG2-glia form with axons at nodes of Ranvier, and the intricate bipartite sheaths formed between the processes of NG2-glia and astrocytes. In addition, we provide evidence that NG2-glia express synaptophysin, indicating they have mechanisms for transmitting as well as receiving signals. This study places NG2-glia within a neuron-glial network, and identifies roles for glutamate and ATP in communication with astrocytes as well as axons.