Vitamin B12 (VB12) is a putative modulator of the human circadian clock, improving entrainability to the 24 h light-dark cycle. The present study was intended to elucidate the mechanism of VB12 action in an animal model. In male rats free-running under constant dim illumination, a single light pulse of 50-1000 lux for 20 min given at circadian time (CT) 20 induced a 0.28 to 1.08 h phase advance and at CT 14 induced a 0.54 to 2.10 h phase delay. A 3 h intracerebroventricular (icv) infusion of 30 nmol VB12 starting 2 h prior to a 20 min 200 lux light pulse significantly amplified phase shifts in comparison with saline-treated or untreated controls. The mean phase advance (1.13 h) was 1.8-fold greater than that of saline-infused controls, whereas the mean phase delay (2.28 h) was 2.9-fold greater. These values were comparable to the maximal phase shifts caused by 1000 lux light pulses in untreated rats. Since the same VB12 treatment alone had failed to induce a phase shift in a previous experiment, these results indicate that VB12 strongly enhanced light pulse-induced phase shifts and thus augmented the entrainability of the circadian clock to light.