Trifluralin, 2,6-dinitro-N,N-dipropyl-4-trifluoromethylaniline, is a 2,6-dinitro herbicide widely used to control annual grasses and broadleaf weeds in agricultural settings. The association between trifluralin use and common cancer incidence was evaluated among 50,127 private and commercial pesticide applicators in the Agricultural Health Study (AHS), a prospective cohort study of licensed pesticide applicators and their spouses in Iowa and North Carolina. Poisson regression was used to examine internal dose–response relationships, while controlling for important lifestyle factors and other agricultural exposures. Two metrics of exposure (lifetime days and intensity-weighted lifetime days) were used in exposure–response analyses with non-exposed applicators, as well as applicators in the lowest tertile of exposure, as reference groups. Incident cancers were identified through state tumor registries from enrollment in 1993 through 2002. Trifluralin exposure was not associated with cancer incidence overall among 51% of private and commercial applicators (n=25,712) who had used trifluralin. However, there was an excess of colon cancer in the exposure category of higher half of highest tertile (rate ratios (RR) of 1.76 (95% CI=1.05–2.95) using the non-exposed as a referent and 1.93 (95% CI=1.08–3.45) using those with the lowest tertile of exposure as the referent). There was also a non-significantly elevated risk for kidney cancer and bladder cancer in the highest exposure group, although only the kidney cancer finding was consistent across exposure metrics. Although there was a possible link between trifluralin exposure and colon cancer, small numbers and inconsistencies in dose–response and subgroup analyses indicate that this may be a chance finding.