Porcine small intestinal submucosa (SIS) is a bioprosthetic collagen material used in the management of various surgical conditions, especially hernia repairs. We studied the effectiveness of porcine SIS Bioprosthetic plug (Surgisis AFP, Cook Biotech Inc., West Lafayette, IN, USA) in the treatment of fistula-in-ano. A prospective multi-institutional study was conducted on 73 patients with anorectal fistulas of differing etiologies. All plugs were inserted in the operating room under anesthesia in patients with preoperative bowel preparation. Regular follow-up was scheduled at 2 weeks, 3, 6, and 12 months. The primary end point was complete closure of the fistula and cessation of drainage over the follow-up period. Seventy-eight AFPs were inserted in 73 patients (28 women and 45 men). Rectovaginal fistulas were excluded. Crohn's disease accounted for 11% (8/73) of the patients. Seventy-three percent of patients (n = 53) had primary fistulas whereas 27% (N = 20) had recurrent fistulas. The plug extrusion (fallout) rate was 9% (7/78). There was no difference in closure rates between primary and recurrent fistulas (primary = 20/53 = 38% and recurrent 8/20 = 40%). The overall patient success rate was 38% (28/73) and the plug success rate was 39.5% when plug fallouts were eliminated. The fistulas in four out of eight patients with Crohn's disease closed (50%). There were no intraoperative complications. There were four postoperative abscesses (4/73; 5%). Use of AFP for treatment of fistula-in-ano is safe and modestly effective in reasonable long-term (15 months) follow-up. This sphincter conserving procedure should be included in the armamentarium of surgeons in the management of transsphincteric or suprasphincteric fistulas.