Objective To investigate the relationship between acupuncture and the autonomic nerve by measuring heart rate variability (HRV) indices before, during and after acupuncture. Methods Ten-minute HRV indices were measured using Actiheart software in 60 patients without heart disease before insertion, during retention and after withdrawal of a needle at the fourth acupuncture treatment. The indices included time-domain indices SD (standard deviation of R-R intervals), rMSSD (root mean square of successive differences in RR intervals) and BPM (beats per minute) and frequency-domain indices LF (low frequency), HF (high frequency) and LF/HF (ratio of low frequency to high frequency). Heart rate variabilities under different conditions were compared using variance analysis. Results The time indices SD and rMSSD increased and BPM decreased after withdrawal of a needle. The frequency indices LF, HF and LF/HF decreased during retention and after withdrawal of a needle. Conclusion Acupuncture affects HRV indices. The changes help to improve the integration of the cardiac autonomic nerve system and inhibit the tension of the sympathetic nerves.