BACKGROUND An anal fistula plug is a sphincter-sparing procedure that uses biological substances to close an anorectal fistula. This study aimed to evaluate the long-term therapeutic effect of an anal fistula plug procedure in patients with trans-sphincteric fistula-in-ano and to determine the risk factors affecting fistula healing. MATERIAL AND METHODS A single-center retrospective study was performed assessing long-term treatment outcomes of patients with low trans-sphincteric anal fistulas who initially underwent anal fistula plug procedures between August 2008 and September 2012. Risk factors affecting fistula healing were identified using univariate and multivariate analyses. RESULTS A total of 135 patients who had low trans-sphincteric anal fistulas and underwent anal fistula plug procedures were analysed. The overall healing rate was 56% (75/135) with a median follow-up time of 8 years (range, 72-121 months). The primary reasons for treatment failure were plug extrusion (n=12, 20%) and surgical site infection (n=9, 15%), occurring within 30 days after surgery. Multiple logistic regression analysis showed that the duration of anal fistula ≥6 months was significantly associated with treatment failure using an anal fistula plug (OR=3.187, 95% CI: 1.361-7.466, P=0.008). Of the patients who failed initial treatment with an anal fistula plug, 6 (9%) had anal fistulas that healed spontaneously after 2-3 years without additional treatment. CONCLUSIONS As a sphincter-preserving procedure, the anal fistula plug can effectively promote healing of low trans-sphincteric anal fistulas. The long-term efficacy is good and the procedure warrants wider use in clinical practice.