Purpose: Infantile hypertrophic pyloric stenosis is the most common cause of gastric outlet obstruction in the first month of life. Botulinum toxin (BT) is a neurotoxin produced by clostridium botulinum, which causes paralysis in skeletal muscles. We aimed to evaluate the effectiveness of BT in the experimental pyloric stenosis model. Methods: The study protocol was approved by the Selcuk University Medical Faculty Ethics Committee (2017/20). We performed an experimental study using 32 Wistar-Albino newborn rats. Rats were divided randomly into four groups with six rats in both control (C), and L-nitro-arginine methyl ester hydrochloride group, and 10 rats in each sham (S), and BT group. 100 mg/kg per day L-NAME was applied to all groups intraperitoneally for 14 days from birth except control group. 0.2 mL saline and 20 U/kg BT was injected by surgery to S and BT groups, respectively, at 21 days from birth. After 35 days all rats were sacrificed and biopsies were performed from pyloric muscle for histopathological examination. The results were evaluated with the "one-way ANOVA"test. Results: Total and circular muscle thickness of the groups were compared. The total muscle thickness of the L-NAME group was significantly higher than the control group (P = .031). Comparing the circular muscle thickness of botox group (BTG) with control group (CG) and L-NAME GROUP (LNG), muscle thickness was significantly smaller (P < .001, P < .001). The total muscle thickness of BTG was significantly different between LNG (P < .001). Conclusions: Hypertrophy of pylor in an experimental model was reduced by BT injection in this study. We think that Botox injection through endoscopic or interventional radiological methods may be an alternative method for surgery.