Structured Abstract Background Prior research suggests surgical safety checklists (SSCs) are associated with reductions in postoperative morbidity and mortality as well as improvement in teamwork and communication. These findings stem from evaluations of individual or small groups of hospitals. Studies with more hospitals have assessed the relationship of checklists with teamwork at a single point in time. The objective of this study was to evaluate the impact of a large-scale implementation of SSCs on staff perceptions of perioperative safety in the operating room. Study Design As part of the Safe Surgery 2015 initiative to implement SSCs in South Carolina hospitals, we administered a validated survey designed to measure perception of multiple dimensions of perioperative safety among clinical operating room personnel before and after implementation of an SSC. Results Thirteen hospitals administered baseline and follow-up surveys, separated by one to two years. Response rates were 48.4% at baseline (929/1921) and 42.7% (815/1909) at follow-up. Results suggest improvement in five of the five dimensions of teamwork (relative percent improvement ranged from +2.9% for coordination to +11.9% for communication). These were significant after adjusting for respondent characteristics, hospital fixed-effects, and multiple comparisons, and clustering robust standard errors by hospital (all p<0.05). More than half of respondents (54.1%) said their surgical teams always used checklists effectively; 73.6% said checklists had averted problems or complications. Conclusions A large-scale initiative to implement SSCs is associated with improved staff perceptions of mutual respect, clinical leadership, assertiveness on behalf of safety, team coordination and communication, safe practice, and perceived checklist outcomes.