Orexin neurons are involved in homeostatic regulatory processes, including arousal and feeding, and provide a major input from the hypothalamus to the ventral tegmental area (VTA) of the midbrain. VTA neurons are a central hub processing reward and motivation and target the medial prefrontal cortex (mPFC) and the shell part of nucleus accumbens (NAcs). We investigated whether subpopulations of dopamine (DA) neurons in the VTA projecting either to the mPFC or the medial division of shell part of nucleus accumbens (mNAcs) receive differential input from orexin neurons and whether orexin exerts differential electrophysiological effects upon these cells. VTA neurons projecting to the mPFC or the mNAcs were traced retrogradely by Cav2-Cre virus and identified by expression of yellow fluorescent protein (YFP). Immunocytochemical analysis showed that a higher proportion of all orexin-innervated DA neurons projected to the mNAcs (34.5%) than to the mPFC (5.2%). Of all sampled VTA neurons projecting either to the mPFC or mNAcs, the dopaminergic (68.3 vs. 79.6%) and orexin-innervated DA neurons (68.9 vs. 64.4%) represented the major phenotype. Whole-cell current clamp recordings were obtained from fluorescently labeled neurons in slices during baseline periods and bath application of orexin A. Orexin similarly increased the firing rate of VTA dopamine neurons projecting to mNAcs (1.99 ± 0.61 Hz to 2.53 ± 0.72 Hz) and mPFC (0.40 ± 0.22 Hz to 1.45 ± 0.56 Hz). Thus, the hypothalamic orexin system targets mNAcs and to a lesser extent mPFC-projecting dopaminergic neurons of the VTA and exerts facilitatory effects on both clusters of dopamine neurons.