To observe the effect of electroacupuncture (EA) on swallowing and its underlying mechanism, 32 Sprague-Dawley (SD) rats were chose and the electrophysiology was used to detect the discharge of nucleus ambiguus (NA) after EA at CV23 (Lianquan), GV16 (Fengfu), and other acupoints. The swallowing-related motor neuron was identified by antidromic stimulation through recurrent laryngeal nerve. Meanwhile, the swallowing numbers were induced by Double-distilled water (DDW) and the neuron discharges were recorded before and after EA. Beside, 50 SD rats were used for testing the c-fos expressions in NA after EA at different acupoints and the other 80 SD rats were used for chemical damage through the microinjection to bilateral NA. 58 neurons provided complete data after histological identification. And two types of swallowing-related (SR) motor neurons were identified, named spontaneous and silent neurons. We found that the onset latency of the first swallow was shorter and the swallowing numbers were increase d after EA at CV23 than the other acupoints (P < 0.01). The excitatory neuron response rates were 66.67%, 71.11%, 42.22% and 35.56% for CV23, GV16, PC6 (Neiguan), and ST36 (Zusanli), respectively. The c-fos expressions on CV23 and GV16 groups were significantly higher than the other groups (P < 0.05). After chemical damage, the swallowing numbers could not be regulated by EA, but could be regulated by EA after fake damage. The results of the present study demonstrate that EA at CV23 and GV16 could regulate swallowing function via activating swallowing-related motor neurons in NA.