ImportancePatient safety interventions, like the World Health Organization Surgical Safety Checklist, require effective implementation strategies to achieve meaningful results. Institutions with underperforming checklists require evidence-based guidance for reimplementing these practices to maximize their impact on patient safety.ObjectiveTo assess the ability of a comprehensive system of safety checklist reimplementation to change behavior, enhance safety culture, and improve outcomes for surgical patients.Design, Setting, and ParticipantsThis prospective type 2 hybrid implementation-effectiveness study took place at 2 large academic referral centers in Singapore. All operations performed at either hospital were eligible for observation. Surveys were distributed to all operating room staff.InterventionThe study team developed a comprehensive surgical safety checklist reimplementation package based on the Exploration, Preparation, Implementation, Sustainment framework. Best practices from implementation science and human factors engineering were combined to redesign the checklist. The revised instrument was reimplemented in November 2021.Main Outcomes and MeasuresImplementation outcomes included penetration and fidelity. The primary effectiveness outcome was team performance, assessed by trained observers using the Oxford Non-Technical Skills (NOTECH) system before and after reimplementation. The Agency for Healthcare Research and Quality Hospital Survey on Patient Safety Culture was used to assess safety culture and observers tracked device-related interruptions (DRIs). Patient safety events, near-miss events, 30-day mortality, and serious complications were tracked for exploratory analyses.ResultsObservers captured 252 cases (161 baseline and 91 end point). Penetration of the checklist was excellent at both time points, but there were significant improvements in all measures of fidelity after reimplementation. Mean NOTECHS scores increased from 37.1 to 42.4 points (4.3 point adjusted increase; 95% CI, 2.9-5.7; P < .001). DRIs decreased by 86.5% (95% CI, −22.1% to −97.8%; P = .03). Significant improvements were noted in 9 of 12 composite areas on culture of safety surveys. Exploratory analyses suggested reductions in patient safety events, mortality, and serious complications.Conclusions and RelevanceComprehensive reimplementation of an established checklist intervention can meaningfully improve team behavior, safety culture, patient safety, and patient outcomes. Future efforts will expand the reach of this system by testing a structured guidebook coupled with light-touch implementation guidance in a variety of settings.