Abstract Pelvic pain persists despite standard care in about 30% of women with a history of endometriosis and pain. Pelvic pain may be accompanied by spasm in pelvic floor muscles that results in sexual pain, impairs intimacy, and might contribute to maintenance of chronic pelvic pain. These women also often have pain sensitization, within and beyond the pelvis. Botulinum toxin is commonly used for conditions with muscle overactivity and pain. We therefore evaluated its potential to decrease pelvic floor spasm and ameliorate chronic pelvic pain in such women. In a randomized, double-masked, placebo-controlled trial, women aged 18-50 years with surgically diagnosed endometriosis and chronic pelvic pain despite optimized hormonal treatment received 100U onabotulinumtoxinA or placebo into pelvic floor muscles with palpable spasm (NCT01553201). At least one month after the masked injection and any time over the next year, participants could request a 2nd injection, which was always botulinum toxin, again injected into muscles with spasm. Pain and spasm were assessed before the first (masked) study injection and one month later (before a second open injection of botulinum toxin) as the primary outcome measure and then at regular intervals over the next year, including after the optional 2nd injection. The women were able to continue their regular pain medications while in the study but were asked not to change any hormonal treatments they were using. Pain medication usage and other aspects of response, including side effects, were tracked. This National Institutes of Health study included 29 women who were premenopausal and who had persistent pain for over 10 years on average despite having optimized standard treatment, such as surgery and hormones, for their endometriosis. All had pelvic floor muscle spasm and described the pelvic floor as a major focus of pain. Twenty-two of 23 reporting current hormone use had menses suppression. Nineteen of 29 characterized pelvic tenderness as focal, not diffuse. All showed widespread myofascial dysfunction with myofascial trigger-points in >two-thirds of assessed regions. Widespread sensitization was present in 16/29; regional sensitization (thoracic: 20/29; pelvic 17/29) was common. The primary outcome, participants’ self-report of benefit and pain relief, showed that botulinum toxin was more likely than placebo to provide benefit. On exam, the women receiving toxin had a corresponding decrease in spasm in the muscles of the pelvic floor compared to before injection. Twenty-seven women requested a 2nd injection, 16 at the one-month time point and 11 later in the year. The 2 patients who did not ask for a 2nd injection had received botulinum toxin and experienced persistent benefit; one subsequently became pregnant. Over the entire year-long study, only 4 patients, two from the group who initially received botulinum toxin and two from the placebo group, did not have durable benefit even after the 2nd injection. Sixteen women had continued benefit at the end of the study. While there was local pain and discomfort with the intravaginal injections, there were no serious side effects attributable to the botulinum toxin itself at the 100U dose used in the study. Baseline and current disability predicted improvement in pain, sexual intercourse, functioning, fatigue and mental health scores. We demonstrate pelvic floor muscle spasm as part of endometriosis-associated chronic pelvic pain and relief of pain/spasm from onabotulinumtoxinA compared to placebo that persisted over time. OnabotulinumtoxinA was well-tolerated. The findings illustrate a viscero-somatic reflex relationship between endometriosis lesions and muscle spasm and suggest that pelvic floor spasm may initiate or maintain sensitization. Although additional research is needed, this study supports that onabotulinumtoxinA can safely and effectively treat chronic pelvic pain in women with a history of endometriosis and ongoing spasm in the pelvic floor muscles.