Acupuncture is helpful in treating various diseases, including autonomic nervous system (ANS) dysfunction caused by mental stress. On the other hand, the frontal lobe is suggested to play an important role in stress responses by modulating the ANS. The aim of the study was to evaluate the effects of acupuncture on ANS and frontal lobe activities. We investigated 18 normal adults. We measured the activity of prefrontal cortex (PFC) caused by real acupuncture (WHO-LI4) and sham acupuncture, employing optical topography. To evaluate ANS function, we analyzed heart rate variability (HRV). Analysis of HRV revealed a decrease of the LF/HF ratio, and an increase of the HF power by real acupuncture, indicating a shift to parasympathetic dominancy. Acupuncture also caused cerebral blood oxygenation changes in both directions, that is, an increase and/or a decrease of oxyhemoglobin (Oxy-Hb) in the bilateral PFC. However, the Oxy-Hb change was not correlated with HRV parameters in the majority of cases. One of the possible explanations of the poor correlations might be that the PFC activity induced by acupuncture is not closely linked with ANS function.