Objective Pupillary light reflex test has been considered as a good marker for diabetic autonomic neuropathy. In this study we have evaluated the efficacy of pupillary light reflex test in the detection of diabetic neuropathy. Methods A total of 554 (382 male, 172 female) patients were recruited in this study. The pupillary light reflex test was performed using a portable infrared video-pupillography system (Iriscorder Dual C10641®; Hamamatsu Photonics). The measured parameters were D1, initial diameter before light stimulus (mm); D2, minimum diameter after light (mm); CR, constriction ratio [CR = (D1~D2)/D1]; VC, the maximum velocity of constriction (mm/s). Results Mean age was 61, mean duration of diabetes was 11.1 years. CR was significantly associated with coefficient of variation of CV R-R (P < 0.01), however, CR showed no association with the frequency of orthostatic hypotension. On the other hand, VC was significantly associated with both of CV R-R and orthostatic hypotension. Accordingly VC may represent the autonomic neuropathy more precisely than CR. Actually VC showed significant correlation with vibration perception threshold (P < 0.01). The determined VC which corresponds to the lower limit of normal sensation of vibration was 3.52. The frequency of abnormal Achilles tendon reflex in group of less than 3.6 VC was more than group of over 3.6 VC (P < 0.01). Conclusion Since somatic nerve dysfunction progresses in parallel with the development of autonomic neuropathy, the cut-off point of 3.6 VC may represent the detection limit of autonomic neuropathy. These results suggest that VC may be a useful marker for diagnosis of diabetic polyneuropathy.