Purpose: Ocular trauma leads to loss of corneal clarity resulting in vision deficits. Autophagy plays a critical role in the extracellular matrix, tissue repair, and homeostasis but its precise mechanistic role in regulating corneal function remains unknown. The present study investigated the modulation of autophagy-related genes (LC3, Beclin1, Sqstm1/p62, and Lamp1) in healthy and injured canine corneal stromal fibroblasts (CSFs). Methods: Primary CSFs were generated from healthy donor canine corneas and grown in minimum essential medium. Following incubation, cultures were exposed to nitrogen mustard (NM) and subjected to an autophagy activator, rapamycin (R), or vehicle treatment. Phase-contrast microscopy, quantitative reverse transcription polymerase chain reaction (qRT-PCR), and immunofluorescence staining were used to study the role of autophagy genes in canine corneal wound healing in vitro. Results: Phase-contrast microscopy showed that NM exposure led to morphological changes with stress fibers in CSFs, which was noticeably decreased by rapamycin treatment. Treatment of CSFs with rapamycin alone showed fibroblast hypertrophy while vehicle-treated population of CSFs exhibited typical spindle morphology. The qRT-PCR showed increased expression of LC3, Beclin1, and Lamp1 mRNA when treated with NM, NM+R, and R in comparison to vehicle-treated CSFs. Sqstm1/p62 expression was upregulated in the NM and NM+R treatment groups but was reduced in the R-treated group. Immunofluorescence showed similar results of the protein levels. Conclusions: This study suggests that autophagy is an essential component in corneal healing post-injury. Further, results suggest that targeting autophagy may offer an attractive treatment option to reestablish corneal clarity following ocular insult.