Dysfunctional dopaminergic neurotransmission is central to movement disorders and mental diseases. The dopamine transporter (DAT) regulates extracellular dopamine levels but the genetic and mechanistic link between DAT function and dopamine-related pathologies is not clear. Particularly, the pathophysiological significance of monoallelic missense mutations in DAT is unknown. Here we use clinical information, neuroimaging, and large-scale exome sequencing data to uncover the occurrence and phenotypic spectrum of a novel DAT coding variant, DAT-K619N, which localizes to the critical C-terminal PDZ-binding motif of human (h)DAT. We identified the rare, but recurrent hDAT-K619N variant in exome-sequenced samples of patients with neuropsychiatric diseases and a patient with early-onset, neurodegenerative parkinsonism and comorbid neuropsychiatric disease. We show that hDAT-K619N displays reduced uptake capacity, decreased surface expression, and accelerated turnover in cell cultures. Unilateral expression in mouse nigrostriatal neurons revealed differential effects of hDAT-K619N and hDAT-WT on dopamine-directed behaviors, and hDAT-K619N expression in Drosophila leads to impairments in dopamine transmission with accompanying hyperlocomotion and age-dependent disturbances of the negative geotactic response. Moreover, cellular studies and viral expression of hDAT-K619N in mice demonstrated a dominant-negative effect of the hDAT-K619N mutant. Summarized, our results suggest that hDAT-K619N can effectuate dopamine dysfunction of pathological relevance in a dominant-negative manner.