Dopaminergic neurons regulate and organize numerous important behavioral processes including motor activity. Consistently, manipulation of brain dopamine concentrations changes animal activity levels. Dopamine is synthesized by several neuronal populations in the brain. This study was carried out to directly test whether selective activation of dopamine neurons in the midbrain induces hyperactivity. A pharmacogenetic approach was used to activate midbrain dopamine neurons, and behavioral assays were conducted to determine the effects on mouse activity levels. Transgenic expression of the evolved hM3Dq receptor was achieved by infusing Cre-inducible AAV viral vectors into the midbrain of DAT-Cre mice. Neurons were excited by injecting the hM3Dq ligand clozapine-N-oxide (CNO). Mouse locomotor activity was measured in an open field. The results showed that CNO selectively activated midbrain dopaminergic neurons and induced hyperactivity in a dose-dependent manner, supporting the idea that these neurons play an important role in regulating motor activity.