Background Despite the widely known benefits of physical activity, people with disabilities are more likely to be inactive when compared to people without disabilities. Previous questionnaires that measure barriers physical activity for people with disabilities do not measure barriers from an ecological perspective. Objective The purpose of this study was to develop the Barriers to Physical Activity Questionnaire for People with Mobility Impairments (BPAQ-MI) that measures barriers using an ecological framework. Methods This study consisted of two phases. In Phase one, developed the content validity by (a) developing an item bank, (b) identifying missing items and combining items using a Delphi panel, and (c) refine item wording via cognitive interviews. In Phase two, people with mobility impairments took part in in-person interviews to establish test-retest reliability, internal consistency, and construct validity of the BPAQ-MI. Results Exploratory factor analysis revealed the BPAQ-MI was comprised of eight subscales or factors: health; beliefs and attitudes; family; friends; fitness center built environment; staff and policy; community built environment; and safety. The BPAQ-MI demonstrated very good test-retest reliability. Cronbach's alpha ranged from 0.792 to 0.935. The BPAQ-MI showed significant negative correlations with exercise (minutes/week) and significant positive correlations between BPAQ-MI subscales and inactivity (hours/day). Conclusions The BPAQ-MI is the first questionnaire that places greater equity at measuring barriers to physical activity across the intrapersonal, interpersonal, organizational, and community domains. The BPAQ-MI has the potential to assist researchers in understanding the complex relationship between barriers and ultimately develop physical activity interventions that address these barriers.