Purpose: The purpose of this study was to examine physical activity (PA)/fitness knowledge and its association with PA and sedentary behavior in youth. Method: Eighth grade students from five schools (N = 660) in a midwestern state completed a PE Metrics written test and the Youth Activity Profile to assess PA/fitness knowledge, PA (at school and after school) and sedentary behavior, respectively. Results: Participants were clustered into high, medium, and low knowledge groups. Students in the high knowledge group reported higher level of PA after school (p < 0.05, d = 0.28) but lower level of sedentary behavior than the low knowledge group (p = 0.001, d = -0.45). The low knowledge group also reported higher PA at school (p < 0.05, d = 0.25). PA/Fitness knowledge significantly predicted sedentary behavior, particularly in the low knowledge group (ß = -0.32, t = -2.46, p < 0.05, R2 = 0.105), after controlling for gender and race/ethnicity. Conclusion: Physical education focused on conveying PA/fitness knowledge is warranted to educate youth to move more and sit less.