Background The introduction of ‘Freedom to Speak Up Guardians’ into every NHS trust in England was intended to support workers and trusts to better raise, respond to and learn from speaking-up concerns. However, only broad guidance was provided on how to implement the role. As a result, there is the potential for important local differences to emerge as the role is implemented across England. Objectives The overall aim of this study was to better understand the implementation of Guardians in acute trusts and mental health trusts. Design The Freedom to Speak Up Guardian role was conceptualised as a complex intervention consisting of several interacting and interlocking components spanning the macro level (national organisations), the meso level (individual trusts) and the micro level (employees, teams and wards/units). A mixed-methods study was designed, which consisted of three work packages: (1) a systematic narrative review of the international literature regarding interventions promoting ‘speaking up’ by health-care employees; (2) semistructured telephone interviews with Guardians working in acute hospital trusts and mental health trusts; and (3) qualitative case studies of Freedom to Speak Up Guardian implementation, consisting of observations and interviews undertaken in four acute trusts and two mental health trusts. Interviews were also undertaken with national stakeholders. Setting Acute trusts and mental health NHS trusts in England. Participants Work package 2: Freedom to Speak Up Guardians (n = 87) were interviewed. Work package 3: 116 interviews with key stakeholders involved in pre-implementation and early implementation decision-making, workers who had spoken up to the Guardian, and national stakeholders. Results Wide variability was identified in how the Guardian role had been implemented, resourced and deployed by NHS trusts. ‘Freedom to Speak Up Guardian’ is best considered an umbrella term, and multiple versions of the role exist simultaneously across England. Any comparisons of Guardians’ effectiveness are likely to be possible or meaningful only when this variability is properly accounted for. Many Freedom to Speak Up Guardians identified how a lack of available resources, especially time scarcity, negatively and significantly affected their ability to effectively respond to concerns; their opportunities to collect, analyse and learn from speaking-up data; and, more generally, the extent to which they developed their role and speak-up culture. Limitations It is possible that those whom we interviewed were more receptive of Freedom to Speak Up Guardians or may have been biased by ‘socially desirability’, and their answers may not always have represented respondents’ true perceptions. Conclusions Optimal implementation of the Guardian role has five components: (1) establishing an early, collaborative and coherent strategy congruent with the values of Freedom to Speak Up fosters the implementation of (2) policies and robust, yet supportive, practices (3) informed by frequent and reflexive monitoring of Freedom to Speak Up implementation that is (4) underpinned by sufficient time and resource allocation that leads to (5) a positive implementation climate that is congruent with Freedom to Speak Up values and is well placed to engender positive and sustainable Freedom to Speak Up culture and the well-being of a Guardian. Future work The following recommendations for future research are considered to be of equal priority. Studies of the speaking-up experiences of minority communities and ‘seldom-heard’ workforce groups are a priority requirement. There is also value in undertaking a similar study in non-hospital settings and where peripatetic working is commonplace, such as in ambulance services and in primary care settings. The role of human resources and ‘middle managers’ in the management of concerns is an area requiring further research, especially regarding concerns relating to unprofessional and transgressive behaviours. Devolved administrations in Scotland and Wales have adopted different approaches to speaking up; research undertaken in these contexts would offer valuable comparative insights. Researching the Guardian role ≥ 5 years post implementation is recommended to understand the medium-term impact and the longer-term sustainability of the role and well-being of Guardians. Study registration This study is registered as ISRCTN38163690 and has the study registration CRD42018106311. Funding This project was funded by the National Institute for Health and Care Research (NIHR) Health and Social Care Delivery Research programme and will be published in full in Health and Social Care Delivery Research; Vol. 10, No. 23. See the NIHR Journals Library website for further project information.