The prelimbic cortex (PL) and infralimbic cortex (IL) play a role in context-induced reinstatement of heroin seeking in an animal model of drug relapse. Both the PL and IL receive direct glutamatergic projections from the ventral CA1 (vCA1), which is also involved in context-induced reinstatement of cocaine and heroin seeking. Here we studied the role of vCA1-PL and vCA1-IL projections in context-induced reinstatement of heroin seeking by using electrophysiological, neuropharmacological, chemogenetic, and molecular methods. We showed that context-induced reinstatement of heroin seeking caused selective activation of the vCA1-IL but not vCA1-PL glutamatergic projections, decreased synaptosomal GluA2 expression in the IL, impaired basal synaptic transmission, and facilitation of long-term depression (LTD) in the vCA1-IL pathway. Additionally, chemogenetic inactivation of the vCA1-IL but not vCA1-PL pathway decreased context-induced reinstatement of heroin seeking. Inactivation of the vCA1-IL pathway also reversed synaptosomal GluA2 downregulation and basal transmission reduction, and blocked LTD induction. Taken together, our results demonstrate a critical role of the vCA1-IL glutamatergic projection in context-induced reinstatement of heroin seeking in a rat model of drug relapse.Neuropsychopharmacology advance online publication, 20 December; doi:10.1038/npp.2017.279.