Acupuncture has been reported to affect the autonomic system. Currently, there are no systematic reviews examining the effect of acupuncture on HRV available in the literature. Therefore, the aim of this systematic review was to summarize and critically assess the effects of acupuncture on heart rate variability. We searched the literature using 14 databases for articles published from the earliest available publications until October 2009 without language restrictions. We included randomized clinical trials (RCTs) comparing acupuncture and sham acupuncture. The risk of bias in each study was assessed using the Cochrane criteria. Twelve RCTs met all of the inclusion criteria. One RCT evaluated the effects of acupuncture in patients with minor depression or anxiety disorders and another RCT examined the effect of acupuncture on migraine patients. Another four RCTs tested the effects of acupuncture in healthy subjects who were exposed to several conditions, including mental stress, fatigue from driving, and caffeine intake. The remaining six RCTs assessed the effects of acupuncture on healthy subjects in a normal state without any stressors. Five RCTs found significant differences in HRV between patients treated with acupuncture versus those treated with sham acupuncture (controls). However, the majority of the other RCTs showed inconsistent results or did not identify significant differences in HRV spectral parameters among individuals treated with acupuncture as compared to those treated with sham acupuncture. In conclusion, sham-controlled RCTs showed variable results and no clear evidence that acupuncture has any specific effects on HRV. Therefore, more rigorous research appears to be warranted.