Cholinergic projections from the medial habenula (MHb) to the interpeduncular nucleus (IPN) have been studied for their complex contributions to nicotine addiction and have been implicated in nicotine reinforcement, aversion, and withdrawal. While it has been established that MHb cholinergic projections co-release glutamate, no direct evidence has demonstrated a role for this specific glutamate projection in nicotine consumption. In the present study, a novel floxed Slc17a7 (VGLUT1) mouse was generated and used to create conditional knockout (cKO) mice that lack VGLUT1 in MHb cholinergic neurons. Histochemical approaches and optogenetics-assisted electrophysiology were used to validate the disruption of VGLUT1 from cholinergic MHb to IPN projections. The mice displayed no gross phenotypic abnormalities and exhibited normal exploratory and locomotor behavior in the open-field assay. However, the loss of VGLUT1-mediated glutamate co-release led to increased nicotine self-administration. These findings indicate that glutamate co-release from ventral MHb cholinergic neurons opposes nicotine consumption and provide additional support for targeting this synapse to develop potential treatments to nicotine addiction.