© 2016 Human Kinetics, Inc. Background: Prolonged sitting time is associated with numerous health outcomes; however, the role of cardiorespiratory fitness (CRF) in these relationships is largely unexplored. The cross-sectional association between reported sitting time and measured CRF was examined in a large study of healthy men and women. Methods: The analytic sample included 4658 men and 1737 women enrolled in the Cooper Center Longitudinal Study. Unadjusted and adjusted multivariable linear regression models were constructed to examine the association between sitting time and CRF, stratified by sex and meeting (or not) meeting physical activity (PA) guidelines. Results: In men, CRF was not associated with sitting time after adjustment for potential confounders. In contrast, for women, after adjustment there was a significant association between increased sitting time and lower CRF (P for trend <.001). When stratified by meeting or not meeting PA guidelines, there was no association between sitting time and CRF in men. In women, this relationship was statistically significant regardless of PA category (both P for trend <.05). Conclusions: These results suggest that the association between sitting time and CRF varies by sex. Further, meeting PA guidelines does not appear to modify this relation in either sex.