A nighttime nap is expected to mitigate melatonin suppression during night work by blocking light input to the retina, but it is unclear. In the present study, we investigated the effects of a nap break on melatonin level, subjective sleepiness, and vigilance performance during simulated night work. Eleven healthy young males (mean ± SD age: 22.2 ± 4.1 years) participated in counterbalanced crossover design experiments with two conditions (nap vs. no nap). The subjects performed 12-hour simulated night work from 21:00 to 9:00 h (illuminance: ~500 lx). Subjects with a nap condition took a nap for 2 hours in a dark room from 3:00, while subjects with a no nap condition continued the simulated night work. The results showed that immediately after the 2-h nap break, the melatonin level at 5:00 h temporarily recovered from light-induced melatonin suppression during the simulated night work but significantly suppressed again at 7:00 and 9:00 h. Subjective alertness and vigilance performance were impaired immediately after the nap break but subsequently enhanced. The results suggest that a single nap break for 2 hours could be a strategy to enhance alertness during the last part of night shift but inadequate for mitigating melatonin suppression.