Vascular pulsation at the optic nerve head (ONH) reflects vessel properties. Reduction in the stimulated retinal vasodilatory capacity has been reported in diabetes, but its relation with vascular pulsation is unknown. Here we report a new retinal imaging system for correlative assessment of ONH vascular pulsation and stimulated retinal vasodilation. Retinal reflectance images were acquired before and during light flicker stimulation to quantify arterial and venous vasodilation (DAR, DVR) in subjects with and without diabetic retinopathy (N = 25). ONH vascular pulsation amplitude and frequency (PA, PF), were quantified by curve fitting of periodic intensity waveforms acquired in retinal vasculature (RV) and ONH tissue (ONHT) regions. The relationships between pulsation metrics, heart rate (HR), intraocular pressure (IOP), and vasodilatory responses were evaluated. Pulsation metrics were not significantly different between regions (p ≥ 0.70). In RV, inter-image variabilities of PA and PF were 10% and 6%, whereas inter-observer variabilities were 7% and 2% respectively. In both regions, PF was correlated with HR (p ≤ 0.001). PA was associated with DAR in both regions (p ≤ 0.03), but only with DVR in RV (p ≤ 0.05). Overall, ONH vascular pulsation was associated with stimulated retinal vasodilation, suggesting diabetes may have concomitant effects on retinal vasculature compliance and neurovascular coupling.