Fear is an emotional mechanism that helps to cope with potential hazards. However, when fear is generalized it becomes maladaptive and represents a core symptom of Post-Traumatic Stress Disorder (PTSD). Converging lines of research show that dysfunction of glutamatergic neurotransmission is a cardinal feature of trauma and stress related disorders such as PTSD. However, the involvement of glutamatergic co-transmission in fear is less well understood. Glutamate is accumulated into synaptic vesicles by vesicular glutamate transporters (VGLUTs). The atypical subtype, VGLUT3 is responsible for the co-transmission of glutamate with acetylcholine (ACh), serotonin (5-HT) or GABA. To understand the involvement of VGLUT3-dependent cotransmission in aversive memories, we used a Pavlovian fear conditioning paradigm in VGLUT3 –/– mice. Our results revealed a higher contextual fear memory in these mice, despite a facilitation of extinction. In addition, the absence of VGLUT3 leads to fear generalization, probably due to a pattern separation deficit. Our study suggests that the VGLUT3 network plays a crucial role in regulating emotional memories. Hence, VGLUT3 is a key player in the processing of aversive memories and therefore a potential therapeutic target in stress-related disorders.