Abstract Background Dark adaptation is an energy-requiring process in the outer retina nourished by the profusely perfused choroid. We hypothesized that variations in choroidal thickness might affect the rate of dark adaptation. Method Cross-sectional, observational study of 42 healthy university students (mean age 25 ± 2.0 years, 29 % men) who were examined using an abbreviated automated dark adaptometry protocol with a 2° diameter stimulus centered 5° above the point of fixation. The early, linear part of the rod-mediated dark adaptation curve was analyzed to extract the time required to reach a sensitivity of 5.0 × 10−3 cd/m2 (time to rod intercept) and the slope (rod adaptation rate). The choroid was imaged using enhanced-depth imaging spectral-domain optical coherence tomography (EDI-OCT). Results The time to the rod intercept was 7.3 ± 0.94 (range 5.1 - 10.2) min. Choroidal thickness 2.5° above the fovea was 348 ± 104 (range 153–534) μm. There was no significant correlation between any of the two measures of rod-mediated dark adaptation and choroidal thickness (time to rod intercept versus choroidal thickness 0.072 (CI95 -0.23 to 0.38) min/100 μm, P = 0.64, adjusted for age and sex). There was no association between the time-to–rod-intercept or the dark adaptation rate and axial length, refraction, gender or age. Conclusion Choroidal thickness, refraction and ocular axial length had no detectable effect on rod-mediated dark adaptation in healthy young subjects. Our results do not support that variations in dark adaptation can be attributed to variations in choroidal thickness.