Axon loss determines persistent disability in multiple sclerosis patients. Here, we use in vivo calcium imaging in a multiple sclerosis model to show that cytoplasmic calcium levels determine the choice between axon loss and survival. We rule out the endoplasmic reticulum, glutamate excitotoxicity, and the reversal of the sodium-calcium exchanger as sources of intra-axonal calcium accumulation and instead identify nanoscale ruptures of the axonal plasma membrane as the critical path of calcium entry. Display omitted •Cytoplasmic calcium accumulations predict axon degeneration in neuroinflammation•Release from the axoplasmic reticulum does not explain calcium accumulations•Calcium influx from the extracellular space drives axon degeneration•Nanoscale ruptures allow entry of calcium across the axonal plasma membrane Witte et al. identify cytoplasmic calcium accumulations as a key driver of axon degeneration in a model of multiple sclerosis. Calcium accumulates in the axoplasm because nanoscale ruptures of the axonal plasma membrane provide an entry path for extracellular calcium.