The COVID-19 pandemic has exacted a physical and mental health toll on health care and hospital workers (HHWs). To provide COVID-19 care, HHWs expected health care institutions to support equipment and resources, ensure safety for patients and providers, and advocate for employees’ needs. Failure to do these acts has been defined as institutional betrayal. Using a mixed-methods approach, this study aimed to explore the experience of institutional betrayal in HHWs serving COVID-19 patients and the associations between self-reported institutional betrayal and both burnout and career choice regret. Between July 2020 and January 2021, HHWs working in an urban U.S. health care system participated in an online survey (n = 1,189) and semistructured interview (n = 67). Among 1,075 quantitative participants, 57.8% endorsed institutional betrayal. Qualitative participants described frustration when the institution did not prioritize their safety while reporting they perceived receiving inadequate compensation from the system and felt that leadership did not sufficiently respond to their needs. Participants who endorsed prolonged breaches of trust reported more burnout and stronger intent to quit their job. Quantitatively, institutional betrayal endorsement was associated with 3-fold higher odds of burnout, aOR = 2.94, 95% CI [2.22, 3.89], and 4-fold higher odds of career choice regret, aOR = 4.31, 95% CI [3.15, 5.89], compared to no endorsement. Developing strategies to prevent, address, and repair institutional betrayal in HHWs may be critical to prevent and reduce burnout and increase motivation to work during and after public health emergencies.