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This study aimed to examine the associations of physical activity and cardiorespiratory fitness (hereafter fitness) with incident glaucoma in a prospective observational study.Physical activity was measured by self-reported leisure-time activities, and fitness was measured by maximal treadmill test. Incident glaucoma was defined based on physician diagnosis. Participants were 9519 men and women between the ages of 40 and 81 yr old (mean age 50 yr) who were enrolled in the Aerobics Center Longitudinal Study. Hazard ratios (HR) were estimated using Cox proportional hazard regression after adjusting for age, sex, race, examination year, smoking status, heavy alcohol drinking, hypertension, hypercholesterolemia, abnormal ECG, diabetes, cardiovascular disease, and cancer.A total of 128 cases of incident glaucoma were reported during a mean follow-up of 5.7 yr. A significantly lower risk of incident glaucoma (HR = 0.53, 95% confidence interval [95% CI] = 0.35–0.79) was found in individuals who met the physical activity guidelines of ≥500 MET·min·wk−1 compared with inactive individuals (0 MET·min·wk−1). Compared with low fitness (lower third), individuals with high fitness (upper third) also had a significantly lower risk of incident glaucoma (HR = 0.60, 95% CI = 0.38–0.95). A joint analysis of physical activity and fitness showed that meeting physical activity guidelines and being in the high fitness category was associated with the lowest risk for developing glaucoma (HR = 0.49, 95% CI = 0.31–0.79).These data provide epidemiological evidence that meeting physical activity guidelines or being fit reduces the risk of developing glaucoma.