Palmer amaranth (Amaranthus palmeri S. Watson) is a troublesome weed in several cropping systems in the US. The evolution of resistance to multiple herbicides is a challenge for the management of this weed. Recently, we reported metabolic resistance to 2,4-D mediated by cytochrome P450 activity in a 6-way-resistant A. palmeri population (KCTR). Plant growth temperature can influence the herbicide efficacy and level of resistance. The effect of temperature on 2,4-D resistance in A. palmeri is unknown. In this research, we investigated the response of KCTR and a known susceptible (MSS) A. palmeri response to 2,4-D grown at low- (LT, 24/14°C, d/n) or high (HT, 34/24°C, d/n) temperature regimes. When MSS and KCTR plants were 8-10 cm tall, they were treated with 0, 140, 280, 560 (field recommended dose), 1120, and 2,240 g ai ha-1 of 2,4-D. Further, 8-10 cm tall MSS and KCTR plants grown at LT and HT, were also treated with [14C] 2,4-D to assess the metabolism of 2,4-D at LT and HT. The results of dose-response experiments suggest that KCTR A. palmeri exhibits 23 times more resistance to 2,4-D at HT than MSS. Nonetheless, at LT, the resistance to 2,4-D in KCTR was only 2-fold higher than MSS. Importantly, at HT there was enhanced metabolism of 2,4-D in both KCTR and MSS A. palmeri, than at LT. Further, treatment with cytochrome P450- inhibitor, malathion followed by 2,4-D increased the susceptibility of KCTR at HT. Overall, rapid metabolism of 2,4-D increased KCTR resistance to 2,4-D at HT compared to LT. Therefore, the application of 2,4-D when temperatures are cooler can improve control of 2,4-D-resistant A. palmeri.