Aim. To summarize all relevant trials and critically evaluate the effect of acupuncture on heart rate variability (HRV). Method. This was a systematic review with meta-analysis. Keyword search was conducted in 7 databases for randomized controlled trials (RCTs). Data extraction and risk of bias were done. Results. Fourteen included studies showed a decreasing effect of acupuncture on low frequency (LF) and low frequency to high frequency ratio (LF/HF ratio) of HRV for nonhealthy subjects and on normalized low frequency (LF norm) for healthy subjects. The overall effect was in favour of the sham/control group for high frequency (HF) in nonhealthy subjects and for normalized high frequency (HF norm) in healthy subjects. Significant decreasing effect on HF and LF/HF ratio of HRV when acupuncture was performed on ST36 among healthy subjects and PC6 among both healthy and nonhealthy subjects, respectively. Discussion. This study partially supports the possible effect of acupuncture in modulating the LF of HRV in both healthy and nonhealthy subjects, while previous review reported that acupuncture did not have any convincing effect on HRV in healthy subjects. More published work is needed in this area to determine if HRV can be an indicator of the therapeutic effect of acupuncture.