The early post-hatching period is the most critical phase in the life of chickens due to its important impact on the physical and functional development of the gastrointestinal tract. The activity of the brain-gastrointestinal axis is regulated by the vagus nerve (acetylcholine, ACh) and hormones modulating rapid growth and development of intestinal cells. Co-regulation of insulin like growth factor 1 (IGF-1) and somatostatin (STS) plays a unique role during extensive cell proliferation. However, the relationship between the cholinergic system and growth factors in the intestine of newly hatched chickens remains unclear. Therefore, an in vitro experiment (6-h tissue culture) was conducted to examine the impact of cholinergic receptor antagonists (atropine and hexamethonium) on IGF-1 and STS secretion in the duodenum of the newly hatched chickens 2 and 24 h after hatching (Day 0 and Day 1). Additionally, the activity of cholinergic muscarinic receptor 3 (CHRM3) in the duodenum was visualised using immunofluorescence. The results showed that the magnitude of the effect of cholinergic receptors inhibition on IGF-1 and STS release and CHRM3 expression depended on the antagonist and time after hatching. In conclusion, the findings clearly demonstrated a connection between the cholinergic system and growth factors in the digestive tract of newly hatched chickens.