© 2016 Elsevier Inc. Purpose To determine the association of different levels of cardiorespiratory fitness (CRF), an objective indicator of habitual physical activity, with gallbladder disease. Methods In the Aerobics Center Longitudinal Study (ACLS) database, 41,528 men and 13,206 women aged 20–90 years, with body mass index of 18.5 or more and without history of cardiovascular disease and cancer, received a preventive examination at the Cooper Clinic in Dallas, Texas, between 1970 and 2003. CRF was quantified as maximal metabolic equivalents and classified as low, moderate, and high based on traditional ACLS cut points. Gallbladder disease was defined as physician-diagnosed gallbladder disease. Results When compared with low CRF, adjusted odds ratios and 95% confidence intervals for gallbladder disease for those with moderate and high CRF were 0.74 (0.55–0.99) and 0.59 (0.42–0.82), respectively when adjusted for all the potential confounders. Each one metabolic equivalent increment of CRF was associated with 10% lower odds of gallbladder disease in all participants (P for trend <.001), 13% lower in women (P for trend <.001), and 8% lower in men (P for trend = .08). The association was consistent across age, history of diabetes mellitus, and physical inactivity subgroups. Conclusions CRF is inversely related to the prevalence of gallbladder disease among relatively healthy men and women in the ACLS cohort.