Primary open angle glaucoma is characterised by the progressive and irreversible death of retinal ganglion cells. Experimental evidence suggests that the initial site of injury to the retinal ganglion cell is at or near the lamina cribrosa or in the peripapillary retina. However, the mediators of axonal injury remain poorly understood. The purpose of this study was to investigate the expression of the gap junction protein connexin43 (GJA1) in the human glaucomatous optic nerve head and retina as a potential mediator of axonal injury. Using affinity isolated polyclonal antibodies to the C-terminal segment of human connexin43, the expression of connexin43 was determined in post-mortem human eyes with primary open angle glaucoma and age-matched controls. In normal eyes, connexin43 was present on glial fibrillary acidic protein (GFAP)-positive astrocytes in the retinal ganglion cell layer and optic nerve head. In glaucomatous eyes, increased connexin43 immunoreactivity was observed at the level of the lamina cribrosa and in the peripapillary and mid-peripheral retina in association with glial activation. This novel finding may suggest that gap junction communication is a potential mediator of retinal ganglion cell injury in glaucoma.