Palmer amaranth (Amaranthus palmeri S. Watson) is a troublesome weed in several cropping systems in the United States. 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 possibly mediated by cytochrome P450 (P450) activity in a six-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 the present research, we investigated the response of KCTR and a known susceptible (MSS) A. palmeri response to 2,4-D grown under low-temperature (LT, 24/14 C, day/night [d/n]) or high-temperature (HT, 34/24 C, d/n) regimes. When MSS and KCTR plants were 8- to 10-cm tall, they were treated with 0, 140, 280, 560 (field recommended dose), 1,120, and 2,240 g ai ha^–1 of 2,4-D. Further, 8- to 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 in MSS. Importantly, there was enhanced metabolism of 2,4-D in both KCTR and MSS A. palmeri at HT compared with LT. Further, treatment with the 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 with LT. Therefore, the application of 2,4-D when temperatures are cooler can improve control of 2,4-D–resistant A. palmeri.