Little is known about the underlying mechanisms governing the bioaccumulation of uranium (U) in aquatic insects. We experimentally parameterized conditional rate constants for aqueous U uptake, dietary U uptake, and U elimination for the aquatic baetid mayfly Neocloeon triangulifer. Results showed that this species accumulates U from both the surrounding water and diet, with waterborne uptake prevailing. Elevated dietary U concentrations decreased feeding rates, presumably by altering food palatability or impairing the mayfly’s digestive processes, or both. Nearly 90% of the accumulated U was eliminated within 24 h after the waterborne exposure ceased, reflecting the desorption of weakly bound U from the insect’s integument. To examine whether the experimentally derived rate constants for N. triangulifer could be generalized to baetid mayflies, mayfly U concentrations were predicted using the water chemistry and U measured in periphyton from springs in Grand Canyon (United States) and were compared to U concentrations in spring-dwelling mayflies. Predicted and observed mayfly U concentrations were in good agreement. Under the modeled site-specific conditions, waterborne U uptake accounted for 52–93% of the bioaccumulated U. U accumulation was limited in these wild populations due to a combination of factors including low concentrations of bioavailable dissolved U species, slow U uptake rates from food, and fast U elimination.