OBJECTIVES: The aims of the study were to systematically review the literature and to synthesize the evidence for the effectiveness of botulinum toxin injection to the pelvic floor muscles for treating pelvic floor myofascial pain in female patients. METHODS: This systematic literature search was performed in February 2018 and updated in September 2019. Articles were screened based on predefined criteria: (1) adult population, (2) female patients, (3) treatment of pelvic pain by transvaginal botulinum toxin injection into the pelvic floor, (4) published in English or English translation available, (5) study design including randomized controlled trials, cohort studies, and case series with more than 10 participants, and (6) quantitative report of pain scores. Nine studies were included in the primary analysis, and an unpublished study was included in a sensitivity analysis. A random effects model with robust variance estimation was used to estimate the pooled mean difference in patient-reported pain scores after botulinum toxin injection. RESULTS: A statistically significant reduction in patient-reported pain scores was noted at 6 weeks after botulinum toxin injection (mean difference, 20.3; 95% confidence interval, 11.7-28.9) and continued past 12 weeks (mean difference, 19.4; 95% confidence interval, 14.6-24.2). Significant improvement was noted in secondary outcomes including dyspareunia, dyschezia, and quality of life. CONCLUSIONS: This systematic review and meta-analysis support the conduct of future, large-scale randomized controlled trials to determine the efficacy and optimize administration of botulinum toxin injections for treatment of pelvic floor myofascial pain and associated symptoms in women.