Glaucoma is characterized by degeneration of optic nerve axons and death of retinal ganglion cells (RGCs). Nerve crush and axotomy of the optic nerve are studied as models of RGC death in glaucoma and of axon regeneration. The mechanisms underlying the response of RGCs to axonal injury remain unclear, however. We have now examined the effects of optic nerve crush on the expression of members of the semaphorin family of neuronal guidance proteins in the rat retina. The expression of semaphorin 3F (Sema3F) in the retina was down-regulated at both the mRNA and protein levels at 7 days after optic nerve injury, whereas that of Sema3A, Sema3B or Sema3C remained unaffected. Immunohistofluorescence analysis and laser capture microdissection followed by reverse transcription–polymerase chain reaction analysis revealed that this loss of Sema3F expression occurred in the RGC layer of the retina. Furthermore, antibody-mediated neutralization of secreted Sema3F in retinal organ culture resulted in down-regulation of neuron-specific βIII-tubulin (Tuj-1 antigen), a marker of RGCs. Our results suggest that Sema3F may contribute to the regulation of RGC function or survival and therefore warrants further investigation as a potential mediator of neuroprotection. Copyright © 2016 John Wiley & Sons, Ltd.