The reduced gait stability and aerobic fitness of people with Down syndrome (DS) may increase their rate of gross oxygen uptake (gross-VO(2)) during over-ground walking. If so, the ACSM equation predicting gross-VO(2) from speed may not be appropriate and an equation specifically for these individuals may be needed. This study therefore examined whether the relationship between gross-VO(2) and speed differs between individuals with and without DS during over-ground walking and attempted to develop a gross-VO(2) prediction equation for people with DS. Gross-VO(2) was measured in 18 persons with DS (24.7 ± 6.8 years; 8 men) and 22 persons without DS (25.9 ± 4.8 years; 9 men) at rest and during five over-ground walking trials, each lasting 6 min, at 0.5, 0.75, 1.0, 1.25, and 1.5 m/s. Multi-level modeling with random intercepts and slopes demonstrated significant effects of speed, group, group-by-speed interaction, and speed squared (P < 0.001). In each group, actual gross-VO(2) did not differ from gross-VO(2) predicted by the regression equation across speeds. Bland-Altman plots showed somewhat greater variability in the difference between actual and predicted gross-VO(2) for participants with DS. Mean absolute error of prediction was 10.75 and 10.67% for participants with and without DS, respectively. In participants with DS, the ACSM formula under-estimated gross-VO(2) across speeds, whereas, in participants without DS, only at 1.5 m/s (P < 0.001). Therefore, individuals with DS show altered curvilinear gross-VO(2) to speed relationship during over-ground walking. The present regression equation appears to offer accurate prediction in people with DS and could be used for prescribing over-ground walking intensities.