Identification of signaling events that contribute to innate spinal cord regeneration in zebrafish can uncover new targets for modulating injury responses of the mammalian central nervous system. Using a chemical screen, we identify JNK signaling as a necessary regulator of glial cell cycling and tissue bridging during spinal cord regeneration in larval zebrafish. With a kinase translocation reporter, we visualize and quantify JNK signaling dynamics at single-cell resolution in glial cell populations in developing larvae and during injury-induced regeneration. Glial JNK signaling is patterned in time and space during development and regeneration, decreasing globally as the tissue matures and increasing in the rostral cord stump upon transection injury. Thus, dynamic and regional regulation of JNK signaling help to direct glial cell behaviors during innate spinal cord regeneration.