Axonal spheroids occur as part of the pathology of a variety of neurologic diseases. Reactive oxygen species (ROS) trigger formation of spheroids, axonal severing, and Ca2+overload. The mechanisms by which ROS lead to the spheroid formation at specific axonal sites remain elusive. Here, using adult mouse primary neurons, we investigate the role of Ca2+, its regulating systems, and cytoskeletal changes in formation of axonal spheroids triggered by ROS. The results reveal that dramatically higher axoplasmic Ca2+levels occur at the sites of axonal spheroids than in the rest of the axon. High focal axoplasmic Ca2+levels correlate with focal aggregation of the reverse Na+/Ca2+exchanger 1, voltage-gated N-type Ca2+channel α1B subunit, and actin at the sites of spheroids in individual axons. This study provides new insights into the mechanism of a spheroid formation at specific sites along axons undergoing oxidative stress and a basis for new neuroprotective strategies.