IMPORTANCE Adverse childhood experiences are common and are associated with changes in early development and learning, but training early childhood educators in trauma-informed approaches to care has not been evaluated with randomized clinical trials. OBJECTIVE To determine whether a 6-session (12-week) professional development course, "Enhancing Trauma Awareness," improved the quality of teachers' relationships with the children in their classrooms. DESIGN, SETTING, AND PARTICIPANTS This cluster randomized clinical trial conducted from September 2017 to May 2018 allocated classrooms by a computer-generated random sequence to intervention (attend Enhancing Trauma Awareness course) and control (no course) groups. Outcomes were reported by participants via survey and analyzed by group allocation. Classrooms under the auspice of the School District of Philadelphia, Pennsylvania, serving 3- and 4-year-old children living in low-income households were invited to participate. Lead and/or assistant teachers from 63 of 348 eligible classrooms (18.1%) agreed to participate, and none were excluded. Of 96 enrolled teachers, 93 (96.9%) were assessed at follow-up (61 of 63 classrooms [96.8%]). EXPOSURES In September 2017, 32 classrooms (48 teachers) were assigned to receive a professional development course that taught about the effects of trauma using a group-based relational process, and 31 classrooms (48 teachers) received no intervention. Teachers completed online surveys immediately before and after the course. Exploratory focus groups with intervention teachers (n = 15) were conducted 5 months after the course ended. MAIN OUTCOMES AND MEASURES The primary outcome was teacher-children relationship quality, with a hypothesized decrease in teacher-children conflict scores. Secondary outcomes included relational capacities (eg, empathy, emotion regulation, and dispositional mindfulness). Focus group themes described teachers' experience of the course. RESULTS Of 96 teachers enrolled, 93 (96.9%) were women, and 58 (60.4%) were 40 years and older. Follow-up surveys were completed by 46 teachers (95.8%) in the control group and 47 (97.9%) in the intervention group, of whom 38 (79.2%) attended 4 or more course sessions. Adjusting for baseline values, mean (SE) conflict scores after the course were not significantly different between course participants (15.8 [0.6]) and controls (15.0 [0.6]) (effect size = 0.16; 95% CI, ?0.19 to 0.52). There were no significant between-group differences in secondary outcomes. However, in focus groups, the teachers reported improvements in teacher-children relationship quality and several related relational capacities. CONCLUSIONS AND RELEVANCE A course to enhance trauma awareness among preschool teachers did not reduce teacher-children conflict scores, yet qualitative assessments suggested the potential for improved teacher-children relationship quality. (author abstract)