Objective: The aim of this study was to evaluate the impact of laser acupuncture on the autonomic nervous system (ANS) of the night shift worker. Background Data: Many articles have demonstrated that levels of affective disorders and stress are high in night shift workers. We applied laser energy to the Neiguan point (PC6) to examine the impact of laser acupuncture on the ANS of 45 healthy young males who were night shift workers and evaluated their heart-rate variability (HRV). Materials and Methods: The laser group (n = 15) received laser acupuncture (9.7 J/cm(2), 830 nm) for 10 min, and the placebo group (n = 15) received sham laser treatment. The effects before and after this intervention on the HRV of the subjects were assessed, along with those seen after 30 min of lying down. Results: After treatment and after the 30-min rest period, the independent-sample t-test showed that both groups exhibited statistically significant differences in high-frequency (HF) HRV, low-frequency (LF) HRV, and the LF: HF ratio of HRV (p < 0.05). Compared with the placebo group, the paired-samples t-test showed that after laser treatment the treatment group had a statistically significant improvement in HF HRV (p = 0.001), LF HRV (p = 0.001), and the LF: HF HRV ratio (p = 0.02). Conclusions: Laser acupuncture stimulation applied to the Neiguan point increased vagal activity and suppression of cardiac sympathetic nerves. This effect was positive and could be used to help patients who have circadian rhythm disorders.