Introduction The persistent development and spread of resistance to antibiotics remains an important public health concern in the UK and globally. About 74% of antibiotics prescribed in England in 2016 was in primary care. The Quality Premium (QP) initiative that rewards Clinical Commissioning Groups (CCGs) financially based on the quality of specific health services commissioned is one of the National Health Service (NHS) England interventions to reduce antimicrobial resistance through reduced prescribing. Emerging evidence suggests a reduction in antibiotic prescribing in primary care practices in the UK following QP initiative. This study aims to investigate the mechanism of impact of this high-cost health-system level intervention on antibiotic prescribing in primary care practices in England. Methods and analysis The study will constitute secondary analyses of antibiotic prescribing data for almost all primary care practices in England from the NHS England Antibiotic Quality Premium Monitoring Dashboard and OpenPrescribing covering the period 2013 to 2018. The primary outcome is the number of antibiotic items per Specific Therapeutic group Age-sex Related Prescribing Unit (STAR-PU) prescribed monthly in each practice or CCG. We will first conduct an interrupted time series using Ordinary Least Square regression method to examine whether antibiotic prescribing rate in England has changed over time, and how such changes, if any, are associated with QP implementation. Single and sequential multiple-mediator models using a unified approach for the natural direct and indirect effects will be conducted to investigate the relationship between QP initiative, the potential mediators and antibiotic prescribing rate with adjustment for practice and CCG characteristics. Ethics and dissemination This study will use secondary data that are anonymised and obtained from studies that have either undergone ethical review or generated data from routine collection systems. Multiple channels will be used in disseminating the findings from this study to academic and non-academic audiences. Strengths and Limitations of this study • This study will be the first to evaluate the mechanism of the impact of a financial incentive initiative involving Clinical Commissioning Groups to improve antibiotic prescribing in primary care practices in England. • The investigation of multiple mediators in this study will help to identify the contributions of multiple strategies in translating the effects of QP while unpacking the extent of the effect of specific mediators. • Due to the limited data on practice-level interventions or strategies that might potentially mediate the effect of the QP on antibiotic prescribing, we will not be able to extensively investigate the mechanism of QP impact at the practice level. • Nevertheless, extensive investigations will be conducted at CCG level where the Quality Premium initiative is implemented, and rewards paid out.