The mechanisms regulating expression of the dopamine transporter are poorly understood. We tested the hypothesis that neuronal activity is one of the non‐genetic determinants of dopamine transporter abundance. Sustained changes in neuronal activity caused by tetrodotoxin and 4‐aminopyridine altered the dopamine uptake and abundance of dopamine transporter and its mRNA in rat mesencephalic cultures. The altered neuronal activity caused by these two drugs is accompanied by changes in intracellular calcium concentrations and Ca²⁺/calmodulin‐dependent protein (CaM) kinase II activity in dopamine neurons. Chronic treatment with an L‐type calcium channel blocker (nifedipine) or CaM kinase inhibitor (KN93) decreased dopamine transporter‐mediated uptake and occluded the effects of tetrodotoxin and 4‐aminopyridine. These data suggest that neuronal activity can regulate dopamine transporter function and abundance via calcium/CaM kinase II signaling.