Background: Children with Hirschsprung's disease may have persistent obstructive symptoms due to internal anal sphincter hypertonicity, even after definitive surgery. Anal myectomy is not universally effective and may result in permanent sphincter injury. Botulinum toxin injection has been used to selectively weaken a variety of muscles and could theoretically represent a less invasive option for children with this difficult problem. We evaluated the efficacy and safety of botulinum toxin in an immature porcine model. Materials and Methods: Six-week-old piglets underwent four-quadrant intrasphincteric injection of botulinum toxin or saline. Under ketamine sedation, internal sphincter resting pressure was measured using a water-perfused system before injection and 4 weeks later. Animals were sacrificed 4 weeks after injection. Histological evidence of neuromuscular changes or inflammation in the internal sphincter was documented. All analysis was done blindly. Results: Internal anal sphincter pressure increased in control animals, likely due to growth over the 4-week period. Conversely, botulinum toxin injection was associated with a significant decrease in internal sphincter pressure. There were no significant differences between the botulinum toxin and control groups with respect to histologic evaluation of neuronal size and number, nerve bundle size and number, inflammation, or muscle atrophy. Conclusions: These data suggest that botulinum toxin is effective in decreasing internal anal sphincter pressure without histologically evident adverse effects. A clinical trial using botulinum toxin for persistent obstructive symptoms following surgery for Hirschsprung's disease is therefore justified.