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The deployment of autonomous shuttles (ASs) holds health and safety benefits for people with and without disabilities. Transportation is critical in helping people with disabilities (PWDs) access health care, services, and jobs, but the current transportation system has not afforded them ubiquitous access. To understand the acceptance of ASs, we (1) quantified PWDs’ (N = 42) perceptions before and after riding in an AS (Level 4) and (2) developed a model of facilitators and barriers from 143 participants. For Objective 1, after riding in the AS, PWDs (n = 42) expressed increased Intention to Use (p < 0.001) and Acceptance (p < 0.001), and decreased Perceived Barriers (p < 0.001), compared with baseline. For Objective 2, four multiple linear regression models were conducted to predict the outcomes for Intention to Use, Perceived Barriers, Well-being, and Acceptance among all participants (N = 143). The results indicated that optimism and ease of use negatively predicted Perceived Barriers and positively predicted Intention to Use, Well-being, and Acceptance. Driving status (i.e., active driver) negatively predicted Intention to Use, Well-being, and Acceptance. Predictors of user Acceptance included optimism, perceived ease of use, driver status, and race/ethnicity—with 30.7% of the variance in Acceptance explained. We also recommended deployment strategies to project stakeholders.