Purpose: The main Purpose: of this study was to investigate the efficacy and safety of botulinum toxin in the treatment of anal fissure. An answer was attempted to the following research questions: (i) what is the efficacy of botulinum toxin in healing of anal fissure compared to placebo, (ii) what is the efficacy of botulinum toxin in healing of anal fissure compared to other means of chemical sphincterotomy, (iii) what is the efficacy of botulinum toxin in healing of anal fissure compared to surgical sphincterotomy, (iv) what is the short term safety of botulinum toxin injections and (v) what is the long term safety of botulinum toxin injections. Methods: Clinical trials investigating the effect of botulinum toxin in the treatment of anal fissure met inclusion criteria. Case reports and case series were also included for the estimation of safety. Meta-analysis was not performed due to clinical heterogeneity. Results: The comparator could be placebo, nitroglycerin ointment, or lateral internal sphincterotomy, with dosage ranging from 20 IU to 50 IU of botulinum toxin. Fissure healing was the most commonly reported primary endpoint but the time period from botulinum toxin injection to fissure healing ranged from 2 weeks to 4 months. Accordingly, outcome data were also heterogenous. Conclusions: Botulinum toxin injections should be considered a minimally invasive therapeutic option for the treatment of chronic anal fissure. However, well designed randomized trials are needed for the valid estimation of the efficacy and safety of botulinum toxin in this therapeutic indication.