The Enlist™ traits provide 2,4-D resistance in several crops. Though corn is naturally tolerant to 2,4-D, the engineered trait conferred by the aryloxyalkanoate dioxygenase-1 (AAD-1) enzyme provides enhanced 2,4-D tolerance and confers resistance to the graminicide herbicide family, the aryloxyphenoxypropionates. The objectives of this research were 2-fold: (1) measure and compare uptake, translocation, and metabolism of 2,4-D in Enlist™ (E, +AAD1) and non–AAD-1 transformed (NT, −AAD1) isogenic corn hybrids; and (2) and investigate the effect of glyphosate and/or the Enlist™ adjuvant system (ADJ) on these factors and corn injury. Uptake of radiolabeled 2,4-D acid applied alone in corn was not altered by the addition of ADJ when tank mixed at 24 h after application (HAA). By contrast, uptake of radiolabeled 2,4-D was significantly lower (69%) compared with 2,4-D plus ADJ (89%) at 24 HAA with a premixed formulation of 2,4-D choline plus glyphosate-dimethylamine (Enlist Duo™ herbicide [EDH]). Translocation of 2,4-D between the two corn hybrids was not different. E corn metabolized more 2,4-D (100% of absorbed) than NT corn (84%), and glyphosate did not alter 2,4-D metabolism. Furthermore, the metabolism of 2,4-D to nonphytotoxic dichlorophenol (DCP) and subsequent DCP-derived metabolites formed in E corn was examined. Injury to E corn is not typically observed in the field; however, injury symptoms were clearly evident in E corn (within 24 HAA) when formulated acetochlor was tank mixed with EDH, which correlated with an increase in 2,4-D uptake during this time period. In summary, the lack of injury in E corn following EDH applied alone may be attributed to a relatively low amount of 2,4-D uptake and the combination of natural and engineered 2,4-D metabolic pathways.