Teamwork training improves short-term teamwork behaviors. However, improvements are often not sustained. The purpose of this study was to explore the extent to which teamwork reinforcement activities for orthopedic surgery teams lead to sustained teamwork behaviors. Seven months after 104 staff from an orthopedic surgical unit were trained in Team Strategies and Tools to Enhance Performance and Patient Safety principles, 4 reinforcement activities were implemented regarding leadership and communication: lectures with videos on leadership skills for nursing staff; an online self-paced learning program on communication skills for nursing staff; a 1-page summary on leadership skills e-mailed to surgical staff; and a 1-hour perioperative grand rounds on Team Strategies and Tools to Enhance Performance and Patient Safety principles for anesthesia staff and new staff. Twenty-four orthopedic surgical teams were evaluated on teamwork behaviors during surgery by 2 observers before and after the reinforcement period using the Observational Teamwork Assessment for Surgery tool. After reinforcement, leadership (P = 0.022) and communication (P = 0.044) behaviors improved compared with prereinforcement levels. Specifically, nursing staff improved in leadership (P = 0.016) and communication (P = 0.028) behaviors, surgical staff improved in leadership behaviors (P = 0.009), but anesthesia staff did not improve in any teamwork behaviors. Sustained improvement in teamwork behaviors requires reinforcement. Level III, prospective pre-post cohort study.