The aim of this study was to evaluate regeneration of a chronic large anal sphincter defect in a pig model after treatment with a plasmid encoding Stromal Cell Derived Factor-1(SDF-1).Under ethics approved protocol 19 age/weight matched Sinclair mini-pigs were subjected to excision of the posterior 50% of anal sphincter muscle and left to recover for 6 weeks. They were randomly allocated to receive either saline treatment (Saline 1 ml, n = 5), 1 injection of SDF-1 plasmid 2 mg/ml (1 SDF-1, n = 9) or 2 injections of SDF-1, 2 mg/ml each at 2 weeks intervals (2 SDF-1, n = 5). Euthanasia occurred 8 weeks after the last treatment. In vivo outcomes included anal resting pressures done under anesthesia pre-injury, pre-injection and before euthanasia (8 weeks after treatment). Anal ultrasound was done pre injury and pre-euthanasia. Tissues were saved for histology and analyzed quantitatively. Two way ANOVA followed by Holm-Sidak test and one way ANOVA followed by the Tukey test were used for data analysis, p < 0.05 was regarded as significant.Posterior anal pressures at the 3 time points were not significantly different in the saline group. In contrast, post-treatment pressures in the 1 SDF-1 group pressures were significantly higher than both pre-injury (p = 0.001) and pre-treatment time points (p = 0.003). At the post-treatment time point, both 1 SDF-1 (p = 0.01) and 2 SDF-1 (p = 0.01) groups had significantly higher mean pressures compared to the saline group. Histology showed distortion of normal anatomy with patchy regeneration in the control group while muscle was more organized in both treatment groups.Eight weeks after a single or two doses of SDF-1injected into a chronic anal sphincter injury improved resting anal pressures and regenerated muscle in the entire defect. SDF-1 plasmid is effective in treating chronic defects of the anal sphincter in a large animal and could be clinically translated.