Objective: Participation in physical activity (PA) among adolescents lowers the risk for various chronic diseases. Yet, few adolescents in the U.S., particularly minority adolescents, meet guidelines of recommended PA (60 minutes daily). Acculturation has been shown to affect PA among adults but is less investigated among Hispanic adolescents. Thus, the objective of this study was to examine the association between acculturation and PA utilizing a nationally representative sample from the 2012 NHANES National Youth Fitness Survey. Methods: A final sample of 119 (weighted sample 3.3 million) adolescents (12–15 years) completed validated acculturation (language as a proxy measure) and PA questionnaires (# of days engaging in 60 minutes/day). Results: After adjustments, and compared to speaking only Spanish/little English, those who spoke equal English/Spanish (β = −0.92, p = 0.05) and more English than Spanish (β = −0.99, p = 0.04) engaged in fewer days of PA. Conclusion: These findings are, generally, in contrast to adult studies, which show that greater acculturation is associated with more PA. Tailored PA and sedentary interventions among Hispanic adolescents should consider their degree of acculturation.