The US overdose epidemic is an escalating public health emergency, accounting for over 100,000 deaths annually. Despite the availability of medications for opioid use disorders, provider-level barriers, such as negative attitudes, exacerbate the treatment gap in clinical care settings. Assessing the prevalence and intensity of provider stigma, defined as the negative perceptions and behaviors that providers embody and enact toward patients with substance use disorders, across providers with different specialties, is critical to expanding the delivery of substance use treatment. To thoroughly understand provider stigma toward patients with substance use disorders, we conducted a nationwide survey of emergency medicine and primary care physicians and dentists using a questionnaire designed to reveal how widely and intensely provider attitudes and stigma can impact these providers' clinical practices in caring for their patients. The survey also queried providers' stigma and clinical practices toward other chronic conditions, which can then be compared with their stigma and practices related to substance use disorders. Our cross-sectional survey was mailed to a nationally representative sample of primary care physicians, emergency medicine physicians, and dentists (N=3011), obtained by American Medical Association and American Dental Association licensees based on specified selection criteria. We oversampled nonmetropolitan practice areas, given the potential differences in provider stigma and available resources in these regions compared with metropolitan areas. Data collection followed a recommended series of contacts with participants per the Dillman Total Design Method, with mixed-modality options offered (email, mail, fax, and phone). A gradually increasing compensation scale (maximum US$250) was implemented to recruit chronic nonresponders and assess the association between requiring higher incentives to participate and providers stigma. The primary outcome, provider stigma, was measured using the Medical Condition Regard Scale, which inquired about participants' views on substance use and other chronic conditions. Additional survey measures included familiarity and social engagement with people with substance use disorders; clinical practices (screening, treating, and referring for a range of chronic conditions); subjective norms and social desirability; knowledge and prior education; and descriptions of their patient populations. Data collection was facilitated through collaboration with the National Opinion Research Center between October 2020 and October 2022. The overall Council of American Survey Research Organizations completion rate was 53.62% (1240/2312.7; physicians overall: 855/1681.9, 50.83% [primary care physicians: 506/1081.3, 46.79%; emergency medicine physicians: 349/599.8, 58.2%]; dentists: 385/627.1, 61.4%). The ineligibility rate among those screened is applied to those not screened, causing denominators to include fractional numbers. Using systematically quantified data on the prevalence and intensity of provider stigma toward substance use disorders in health care, we can provide evidence-based improvement strategies and policies to inform the development and implementation of stigma-reduction interventions for providers to address their perceptions and treatment of substance use. DERR1-10.2196/47548.