The aim of this study was to clarify the effects of transcutaneous vagus nerve stimulation (tVNS) to the left cymba concha on the pain perception using nociceptive withdrawal reflex (NWR), which is known to be associated with chronic pain, and to investigate whether tVNS-induced suppression of the NWR and parasympathetic activation is correlated. We applied either 3.0 mA, 100 Hz tVNS for 120 s in the left cymba concha (tVNS condition) or the left earlobe (Sham condition) for twenty healthy adults. NWR threshold was measured before (Baseline), immediately after (Post 0), 10 min (Post 10) and 30 min after (Post 30) stimulation. The NWR threshold was obtained from biceps femoris muscle by applying electrical stimulation to the sural nerve. During tVNS, electrocardiograph was recorded, and changes in autonomic nervous activity were analyzed. We found that the NWR thresholds at Post 10 and Post 30 increased compared to baseline in the tVNS group (10 min after: p = 0.048 30 min after: p = 0.037). In addition, increased parasympathetic activity by tVNS correlated with a greater increase in NWR threshold at Post 10 and Post 30 (Post 10: p = 0.01; Post 30: p = 0.005). The present results demonstrate the pain-suppressing effect of tVNS as assessed with NWR threshold and suggest that the degree of parasympathetic activation during tVNS may predict the effect of tVNS after its application.