Introduction: Enhancing patient safety and minimizing medical errors are crucial in healthcare. While Root Cause Analysis (RCA) is commonly used to investigate adverse events, its lack of human factors integration limit its effectiveness. The Human Factors Analysis and Classification System (HFACS), adapted from aviation to healthcare, systematically identifies human and organizational factors. However, the integration of HFACS into RCA and the impact of HFACS-RCA implementation remain underexplored. Therefore, this study aims to: provide a practical case of HFACS integration into RCA and explore the effects of HFACS-RCA implementation.Methodology: This study integrates HFACS into the RCA process at a medical center in Taiwan, examining an incident involving unsterilized instruments distributed from the Central Sterile Supply Room (CSR) to the ICU. This study employed a before-and-after study design to examine the impact of the HFACS-RCA intervention. The primary outcome measures were the changes in scores across the eight dimensions of the Taiwan Patient Safety Culture Survey (TPSC) before and after the intervention.Results: A one-year follow-up of the CSR case showed no similar incidents. HFACS-RCA significantly improved TPSC scores in unit safety climate (p=0.05), feelings towards management (p=0.05), and job satisfaction (p=0.05), while the other dimensions showed no significant changes.Conclusion: HFACS-RCA application offers a comprehensive framework for identifying and mitigating factors contributing to medical errors, improving patient safety, and setting a precedent for future healthcare safety management research and practice.