Physical activity (PA) is associated with decreased levels of arterial stiffness in adults, but the relationship between PA and multiple measures of arterial stiffness in adolescents and young adults is not clear. The objective of this study was to test the hypothesis that PA is an independent predictor of multiple measures of arterial stiffness in adolescents and young adults. A total of 548 participants were enrolled in a study of the cardiovascular effects of obesity and type 2 diabetes mellitus (T2DM) (lean, 201; obese, 191; T2DM, 156). Anthropometrics, blood pressure, central and peripheral measures of arterial stiffness (pulse wave velocity, brachial distensibility, and augmentation index), blood (lipids and metabolic tests), and accelerometry data were collected. General linear modeling was performed to test for the independent relationship of PA on arterial stiffness. The mean age of the participants was 17.9 years (standard deviation, 3.5 years). After adjusting for other cardiovascular disease risk factors such as age, sex, body size, mean arterial pressure, and the presence of obesity or T2DM, PA was an independent predictor of augmentation index and brachial distensibility (P < .001). A greater effect of PA on pulse wave velocity was found in participants with T2DM (P = .009) compared with participants in the lean or obese groups. Physical activity is significantly and independently associated with multiple measures of arterial stiffness in adolescents and young adults. The role of PA in the prevention of cardiovascular disease target organ damage in youth, independent of energy balance, merits further exploration.