To clarify the effect of vitamin B12 on the human circadian clock, five healthy male adults participated in two constant routine procedures, 2 or 3 weeks apart. In one, subjects received intravenous saline injections (placebo trial) and in the other, intravenous injections of methylcobalamin (MB12) (drug trial). Intravenous administration of MB12 increased rectal temperature in the later hours of the daytime during the constant routine. The activity did not change between treatments in any period during the constant routine. During the later hours of the constant routine, alertness assessed with visual analog scale was higher in the drug trial than in the placebo trial. The period in which drug treatment produced greater alertness almost coincided with that in which MB12 elevated rectal temperature. These results may provide evidence of an effect of vitamin B12 on the circadian clock.