Amphetamines (AMPHs) are substrates of the dopamine transporter (DAT) and reverse the direction of dopamine (DA) transport. This has been suggested to depend on activation of Ca²⁺-dependent pathways, but the mechanism underlying reverse transport via endogenously expressed DAT is still unclear. Here, to enable concurrent visualization by live imaging of extracellular DA dynamics and cytosolic Ca²⁺ levels, we employ the fluorescent Ca²⁺ sensor jRGECO1a expressed in cultured dopaminergic neurons together with the fluorescent DA sensor GRAB_DA1H expressed in cocultured “sniffer” cells. In the presence of the Na⁺-channel blocker tetrodotoxin to prevent exocytotic DA release, AMPH induced in the cultured neurons a profound dose-dependent efflux of DA that was blocked both by inhibition of DAT with cocaine and by inhibition of the vesicular monoamine transporter-2 with Ro-4-1284 or reserpine. However, the AMPH-induced DA efflux was not accompanied by an increase in cytosolic Ca²⁺ and was unaffected by blockade of voltage-gated calcium channels or chelation of cytosolic Ca²⁺. The independence of cytosolic Ca²⁺ was further supported by activation of N-methyl-D-aspartate-type ionotropic glutamate receptors leading to a marked increase in cytosolic Ca²⁺ without affecting AMPH-induced DA efflux. Curiously, AMPH elicited spontaneous Ca²⁺ spikes upon blockade of the D2 receptor, suggesting that AMPH can regulate intracellular Ca²⁺ in an autoreceptor-dependent manner regardless of the apparent independence of Ca²⁺ for AMPH-induced efflux. We conclude that AMPH-induced DA efflux in dopaminergic neurons does not require cytosolic Ca²⁺ but is strictly dependent on the concerted action of AMPH on both vesicular monoamine transporter-2 and DAT.