The medial septum (MS) impacts hippocampal activity and the hippocampus, in turn, regulates midbrain dopamine (DA) neuron activity. However, it remains to be determined how MS activation impacts midbrain DA activity. This question was addressed by infusing NMDA (0.75 µg/0.2 µL) into the medial septum of anesthetized male Sprague-Dawley rats and recording dopamine neuron activity in the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc). MS activation increased (71%) the number of spontaneously active DA neurons in the VTA, and decreased (40%) the number of active DA neurons in the SNc. Effects in both the VTA and SNc required the ventral subiculum, but were differentially dependent on cholinergic and GABAergic mechanisms within the vSub and rostral and caudal subregions of the ventral pallidum, respectively. MS activation also decreased amphetamine-induced locomotor behavior, which was dependent on GABAergic inputs to the hippocampus. These findings demonstrate that the MS differentially regulates meso-striatal DA transmission via distinct pathways.