AbstractDopamine neurotransmission is highly dysregulated by the psychostimulant methamphetamine, a substrate for the dopamine transporter (DAT). Through interactions with DAT, methamphetamine increases extracellular dopamine levels in the brain, leading to its rewarding and addictive properties. Methamphetamine also interacts with the sigma-1 receptor (σ₁R), an inter-organelle signaling modulator. Using complementary strategies, we identified a novel mechanism for σ₁R regulation of dopamine neurotransmission in response to methamphetamine. We found that σ₁R activation prevents methamphetamine-induced, DAT-mediated increases in firing activity of dopamine neurons. In vitro and in vivo amperometric measurements revealed that σ₁R activation decreases methamphetamine-stimulated dopamine efflux without affecting basal dopamine neurotransmission. Consistent with these findings, σ₁R activation decreases methamphetamine-induced locomotion, motivated behavior, and enhancement of brain reward function. Notably, we revealed that the σ₁R interacts with DAT at or near the plasma membrane and decreases methamphetamine-induced Ca²⁺ signaling, providing potential mechanisms. Broadly, these data provide evidence for σ₁R regulation of dopamine neurotransmission and support the σ₁R as a putative target for the treatment of methamphetamine addiction.