The ubiquitin proteasome system (UPS) plays a vital role in the regulation of protein degradation. Ubiquitination of proteins has been implicated in the pathological cascade associated with neuronal degeneration in both neurodegenerative disease and following acquired central nervous system (CNS) injury. In the present study, we have investigated the role of the UPS following mild to moderate in vitro axonal stretch injury to mature primary cortical neurons, a model of the evolving axonal pathology characteristic of diffuse axonal injury following brain trauma. Transient axonal stretch injury in this model does not involve primary axotomy. However, delayed accumulation of ubiquitin in neuritic swellings at 48 h post-injury (PI) was present in axonal bundles, followed by approximately 60% of axonal bundles progressing to secondary axotomy at 72 h PI. This delayed accumulation of ubiquitin was temporally and spatially associated with cytoskeletal damage. Pharmacological inhibition of the UPS with both MG132 and lactacystin prior to axonal injury resulted in a significant (p < 0.05) increase in the number of axonal bundles progressing to secondary axotomy at 48 and 72 h PI. These results demonstrate that, following mild to moderate transient axonal stretch injury, UPS activity may assist structural reorganization within axons, potentially impeding secondary axotomy. Protein ubiquitination in the axon may therefore have a protective role relative to the diffuse axonal changes that follow traumatic brain injury.