Purpose To study the effect of acute mild hypoxia on retinal oxygen saturation. Methods Spectral retinal images were acquired under normoxic and hypoxic conditions for ten healthy human volunteers (6 male, 4 female, aged (mean±SD) 25±5 years) using a modified fundus camera fitted with an image-replicating imaging spectrometer (IRIS). Acute, mild hypoxia was induced by changing the oxygen saturation of inhaled air from 21% to 15% using a hypoxia generator with subjects breathing the hypoxic gas mixture for 10 minutes. Peripheral arterial oxygen saturation of the subjects was monitored using fingertip-pulse oximetry. Images were processed to calculate oxygen saturation, arteriovenous difference in oxygen saturation and vessel diameter. Data are presented as mean±SD and were analysed using paired sample t-test with significance accepted at P<0.05. Results The retinal arterial and venous oxygen saturation was 98.5±1.6% and 70.7±2.7% during normoxia. A reduction in the fraction of inspired oxygen resulted in a decline (P<0.001) in both retinal arterial and venous oxygen saturation to 90.3±2.0% and 62.4±2.2%, respectively. The arteriovenous oxygen saturation difference in normoxia (27.8±2.9%) and hypoxia (27.9±2.1%) did not change. Retinal arteriolar and venular diameters increased (P<0.001) by 4% and 3% respectively under hypoxia. Conclusions The acute inhalation of a hypoxic gas mixture resulted in a decline in both retinal arterial and venous saturation, whilst arteriovenous oxygen difference was maintained with an accompanying significant increase in retinal vessel diameter. This may suggest an autoregulatory response to acute mild hypoxia.