Central dopamine (DA) innervation of the olfactory tubercle (OT) from the ventral tegmental area (VTA) plays a critical role in encoding multisensory information and generating behavioral outputs necessary for survival. However, due to anatomical restrictions and the neurochemical heterogeneity of the VTA and OT, very little is known about the functional link between mesolimbic VTA-DA transmission in the OT and its role in mediating reward and drug seeking. In this study, we integrated in vivo fast-scan cyclic voltammetry with chemogenetics to (1) characterize the effects of chemogenetic modulation (excitation and inhibition) of mesolimbic DA transmission in the OT of both anesthetized and awake-behaving wild-type rats and (2) demonstrate that inhibition of VTA-DA neurons is sufficient to suppress methamphetamine-induced DA transmission as well as its locomotor and rewarding effects. These results offer novel insights into mesolimbic DA transmission in the OT and its contribution to substance use disorders.