Background: Staple line reinforcement material has been demonstrated to raise the burst pressure threshold after linear intestinal stapling. Numerous bioprosthetic materials have been utilized in surgical practice. Porcine urinary bladder matrix (ACell, Inc.) is an extracellular matrix material derived from porcine bladder used to reinforce surgically repaired soft tissue, and facilitate the body’s regenerative capacity. Objective: This study represents the first evaluation of urinary bladder matrix in gastrointestinal staple line reinforcement. Methods: Pathogen-free pigs underwent midline laparotomy under general anesthesia. Small intestinal division was performed with an endoscopic linear stapler. Nineteen intestinal divisions were performed with urinary bladder matrix staple line reinforcement, and twenty divisions were unreinforced. Staple lines were then subjected to burst pressure analysis by intraluminal infusion of dyed Krebs solution at an infusion rate of 20 ml·min^-1 under manometric monitoring. Upon visible staple line extravasation, intraluminal pressure was recorded. Results: Intestinal staple lines reinforced with urinary bladder matrix exhibited significantly higher burst pressure threshold (p < 0.05). Reinforced staple lines had an average burst pressure of 99 ± 33 mmHg, compared to 61 ± 37 mmHg for unreinforced staple lines. Conclusion: Staple line reinforcement using urinary bladder matrix acutely improves burst pressures of intestinal staple lines when compared with unreinforced staple lines. Its regenerative properties may confer a long-term advantage to staple line reinforcement. These findings, along with previous findings of constructive remodeling in the presence of urinary bladder matrix in treatment of the gastrointestinal system, suggest that UBM may serve a role in gastrointestinal staple line reinforcement.