Background: A common leisure-time activity amongst youth and adults in the United States is video gameplay. Playing video games is typically a sedentary endeavor; however, to encourage an increased level of physical activity in an engaging and enjoyable way, active video gaming has become popular. Unfortunately, the accessibility of gaming controllers is often an issue for persons with disabilities. A commercial off-the-shelf (OTS) gaming mat was adapted to facilitate use by individuals with mobility impairments to address this issue. Objective: Our study aimed to examine energy expenditure, enjoyment, and gameplay experience in youth and adults with mobility impairment during active video gaming using an OTS and adapted versions of a gaming mat. Methods: The study used an observational design. During visit 1, physical function was assessed, and participants were given a familiarization period with the gaming system. For visit 2, based on observation during the physical function tests and discussion with the participant, it was decided whether the participant would play in a standing or seated position. For standing gameplay, the mat was placed on the floor, and for seated play, the mat was placed on a height-adjustable and tilt-adjustable tabletop. Metabolic data were collected during a 20-minute baseline and four 10-minute bouts of Wii Fit Plus gameplay, with 2 bouts on each of the mats (adapted and OTS). During gameplay, the research staff observed and rated participants’ ability to use the game controller (mat) and the quality of gameplay. At the end of each game set, participants reported their rating of perceived exertion on a scale from 0 to 10. During rest, participants completed the physical activity enjoyment scale. Participants also answered additional questions regarding the system's usability with each controller (adapted mat and OTS mat). Statistical analyses were computed using Stata 16 (version 16.1; StataCorp). Linear mixed-effects maximum likelihood regression was performed separately for individuals who could play standing and for those who played seated. Results: A convenience sample of 78 individuals with mobility impairments between the ages of 12 and 60 years (mean 39.6, SD 15.8) participated in the study. Of the sample, 48 participants played the video games in a seated position, while 30 played the games standing. Energy expenditure and heart rate tended to be higher in the OTS mat condition for seated players, while values were similar for both conditions among standing players. However, seated participants reported greater gameplay experience, and both groups exhibited a higher quality of gameplay during the adapted mat condition. Conclusions: Active video gaming using an adapted gaming mat provided an enjoyable exercise activity for individuals with mobility impairments. The use of the adapted controller provides a means by which this population can engage in light to moderate intensity active video gaming, thereby reducing sedentary leisure time.