Uterine fibroids feature excessive deposition of types I and III collagen. Previous ex vivo studies showed an FDA-approved collagenase (EN3835)-digested types I and III collagen fibers in fibroid tissues; however, collagenase had not been evaluated in vivo for effects on uterine fibroids. The objective was to assess the safety and tolerability of collagenase injection directly into uterine fibroids. This was a prospective, open label, dose escalation study. The study participants were fifteen women aged 35-50 years with symptomatic uterine fibroids planning to undergo hysterectomy. Three subjects received saline and methylene blue, three subjects received a fixed dose of EN3835, and 9 subjects received stepped, increasing dosages of EN3835, all by transvaginal, ultrasound-guided injections. Primary outcome measures were safety and tolerability of the injection and change in collagen content between treated and control tissues. There were no significant adverse events following injection of EN3835 into uterine fibroids. Masson's trichrome stains revealed a 39% reduction in collagen content in treated samples compared to controls (p <0.05). Second harmonic generation (SHG) analysis showed treated samples to have a 21% reduction in density of collagen compared to controls. Picrosirius-stained collagenase-treated fibroids showed collagen fibers to be shorter and less dense compared to controls. Subjects reported a decrease in fibroid-related pain on the McGill Pain Questionnaire after study drug injection in Group 2 at both 4-8 days and 60-90 days post-injection. The findings indicated that injection of collagenase was safe and well tolerated. These results support further clinical investigation of collagenase as a minimally invasive treatment of uterine fibroids. NCT0289848.