The aim of this study was to investigate the impact of cigarette smoking in otherwise healthy young individuals on retinal blood flow (RBF) and vascular reactivity (RVR). An automated gas flow controller (RespirAct) was used to achieve normoxic hypercapnia in 10 nonsmokers (mean age 28.9; SD 4.6 years) and nine smokers (mean age 27.55; SD 4.7 years). Retinal blood flow measurements were obtained using a prototype Doppler spectral-domain optical coherence tomographer (SD-OCT) and bidirectional laser Doppler velocimetry and simultaneous vessel densitometry during baseline, normoxic hypercapnia, and recovery. Group mean PETCO2 (end-tidal partial pressure of CO2) was increased by 15.9% in the nonsmoking group and by 15.7% in the smoking group, with a concomitant increase in PETO2 (end-tidal partial pressure of O2) by approximately 1.5% to 2% in both groups. In nonsmokers, retinal arteriolar diameter (P < 0.0001), centerline velocity (P = 0.0004), and blood flow (P < 0.0001) significantly increased during normoxic hypercapnia. Similarly, the venous area (P = 0.0418), venous velocity (P = 0.0068), and total venous RBF (P < 0.0001), as measured by the prototype Doppler SD-OCT, significantly increased. In smokers, normoxic hypercapnia resulted in a significant increase in velocity (P = 0.0019), flow (P = 0.0029), and total venous RBF (P = 0.002). Comparing smokers and nonsmokers, the percentage change in arteriolar diameter (P = 0.0379) and blood flow (P = 0.0101) was significantly lower in the smoking group. There was no significant difference in baseline PETCO2 level between smokers and nonsmokers. Retinal vascular reactivity in response to normoxic hypercapnia is significantly reduced in young, healthy smokers compared with nonsmokers.