To investigate the retinal vascular response to hyperoxia in patients with primary open-angle glaucoma (POAG) using optical coherence tomography angiography (OCTA). This prospective study included 27 eyes in 27 patients with POAG and 14 eyes in 14 age- and sex-matched healthy participants. Retinal radial peripapillary capillary (RPC) perfusion was measured by OCTA before and after inhaling oxygen in all participants. Systemic hemodynamic variables were also examined and recorded before and after hyperoxia. Hyperoxia significantly reduced the perfused vessel density (PVD) of RPCs in both healthy controls (baseline and hyperoxia: 54.2 ± 4.1 and 51.0 ± 4.4, respectively, P < 0.001) and patients with POAG (baseline and hyperoxia: 44.7 ± 6.1 and 43.2 ± 5.4, respectively, P = 0.001). However, the changes in peripapillary PVD between the two gas conditions in patients with POAG were significantly lower than in healthy controls, including both the absolute change (baseline-hyperoxia: 1.5 ± 2.0 and 3.2 ± 1.2, respectively, P = 0.006) and relative change (ratio of absolute change and baseline value: 3.0% ± 4.6% and 6.0% ± 2.4%, respectively, P = 0.04). Retinal microvasculature responds to hyperoxia by reducing RPC perfusion in both healthy participants and patients with POAG. However, this vasoreactivity capacity was significantly impaired in patients with POAG.