This study explored the role of cathepsin B (CTSB) in optic nerve regeneration. Sprague–Dawley rats were utilized for optic nerve crush and long-range crush injury model. Gene and protein expression changes were analyzed via reverse transcription quantitative polymerase chain reaction and western blot. Primary cortical neurons and BV2 cells were cultured to assess CTSB’s effects on neuronal outgrowth and microglial activity. Local CTSB administration degraded chondroitin sulfate proteoglycans (CSPGs), promoting axonal growth in-vivo. In-vitro, CTSB neutralized CSPG-mediated inhibition of neuronal growth. Quantitative proteomics revealed elevated microglial marker proteins in the regenerative environment. Activation of signal transducer and activator of transcription 3 (STAT3) and signal transducer and activator of transcription 6 (STAT6) pathways in BV2 cells increased CTSB secretion. These findings suggest that postinjury regenerative microenvironment reconstruction is associated with upregulated CTSB, which degrades CSPGs to facilitate axonal growth. Microglia-derived CTSB, regulated by STAT3/STAT6 signaling, may play a key role in this process. Modulating CTSB expression could thus be a therapeutic strategy to enhance optic nerve regeneration by modifying the injury microenvironment.