Dynorphin (DYN)/κ-opioid receptor (KOR) activation contributes to aversion, dysphoria, sedation, depression, and enhanced psychostimulant-rewarding effects by inhibiting dopamine (DA) release. The precise neuronal mechanisms underlying these effects remain unclear, limiting the use of KOR agonists in treating mood and substance use disorders. DYN fibers form synapses with DA terminals that express KOR and dopamine transporter (DAT), which is crucial for regulating DA dynamics and related behaviors. Previously, we demonstrated that KOR agonists upregulate DAT activity via ERK1/2 signaling involving phospho-Thr53 DAT (pT53-DAT). However, it remains unclear whether pT53-DAT is involved in KOR-mediated DAT regulation in vivo and whether such a phenomenon contributes to the behavioral effects of KOR agonism. In this study, we utilized male DAT-Ala53 knock-in mice with nonphosphorylatable Ala at position 53 to investigate the role of pT53-DAT in KOR-mediated DAT regulation and its behavioral effects. KOR agonist U69593 increased KOR antagonist-sensitive DAT activity, DAT
Vmax
, pT53-DAT, and surface expression in WT but not in DAT-Ala53 mice. KOR agonists caused locomotor suppression, conditioned place aversion (CPA), and enhanced cocaine preference [conditioned place preference (CPP)] in WT but not in DAT-Ala53 mice. Conversely, both WT and DAT-Ala53 mice exhibited similar lithium chloride-induced CPA and morphine-induced CPP. These findings provide the first causal evidence that KOR-mediated locomotor suppression, aversive response, and enhancement of cocaine reward manifest through the modulation of DAT activity via DAT-Thr53 phosphorylation. This suggests that targeting specific DAT-regulatory motif(s) may help develop new KOR-directed therapeutic strategies devoid of adverse effects.