Aim To examine whether sympathetic afferent stimulation (SAS) inhibits central vagal activation induced by α2‐adrenergic stimulation. Methods In anaesthetized Wistar‐Kyoto rats, a cardiac microdialysis technique was applied to the left ventricle, and the effect of α2‐adrenergic stimulation by medetomidine on myocardial interstitial acetylcholine (ACh) levels was examined in the absence (n = 6) or the presence (n = 6) of SAS delivered from the left stellate ganglion. The effect of electrical vagal efferent stimulation on myocardial interstitial ACh release was also examined in the absence or the presence of SAS (n = 6). Results Intravenous medetomidine (0.1 mg kg−1) significantly increased myocardial interstitial ACh levels in the absence of SAS (from 1.95 ± 0.79 to 3.36 ± 1.61 nm, P < 0.05), but not in the presence of SAS (from 1.67 ± 0.67 to 2.01 ± 0.78 nm). In contrast, electrical vagal nerve stimulation increased myocardial interstitial ACh level to the same degree regardless of SAS (from 1.66 ± 0.16 to 3.93 ± 0.72 nm without SAS vs. 4.05 ± 0.89 nm with SAS). Conclusion Sympathetic afferent stimulation inhibited medetomidine‐induced ACh release, but not electrical stimulation‐induced ACh release, suggesting that SAS inhibited medetomidine‐induced vagal activation via central mechanisms. While central vagal activation by α2‐adrenergic agonists could be an alternative to electrical vagal activation, blocking sympathetic afferent input may be important to increase the efficacy of α2‐adrenergic agonists in enhancing vagal nerve activity.