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Designing and developing safe systems has been a persistent challenge in health care, and in surgical settings in particular. In efforts to promote safety, safety culture, i.e., shared values regarding safety management, is considered a key driver of high-quality, safe healthcare delivery. However, changing organizational culture so that it emphasizes and promotes safety is often an elusive goal. The Safe Surgery Checklist is an innovative tool for improving safety culture and surgical care safety, but evidence about Safe Surgery Checklist effectiveness is mixed. We examined the relationship between changes in management practices and changes in perceived safety culture during implementation of safe surgery checklists. Using a pre-posttest design and survey methods, we evaluated Safe Surgery Checklist implementation in a national sample of 42 general acute care hospitals in a leading hospital network. We measured perceived management practices among managers (n = 99) using the World Management Survey. We measured perceived preoperative safety and safety culture among clinical operating room personnel (N = 2,380 (2016); N = 1,433 (2017)) using the Safe Surgical Practice Survey. We collected data in two consecutive years. Multivariable linear regression analysis demonstrated a significant relationship between changes in management practices and overall safety culture and perceived teamwork following Safe Surgery Checklist implementation.