The habenula is a phylogenetically conserved bilateral brain structure known to modulate negative emotions. Hemispheric asymmetry in cortical areas plays a key role in behavior and cognition. Although habenular asymmetry is prominent in several vertebrate species, there is little evidence for left-right asymmetry in the mammalian habenula. To investigate the asymmetry in synaptic transmission in the medial habenula (MHb) to the interpeduncular nucleus (IPN) pathway in mice, we performed targeted electrical stimulation of left or right MHb-derived axons in acute slices. We discovered that the probability of neurotransmitter release from left MHb terminals was significantly lower than that of right MHb terminals. Furthermore, presynaptic GABAB receptor (GBR) activation with the agonist baclofen more effectively enhanced release from left MHb terminals than from the right.To examine asymmetrical function of this pathway in fear memory, we unilaterally injected Cre-dependent inhibitory DREADDs-expressing adeno-associated virus into choline acetyltransferase-Cre mice and performed cued fear conditioning. Chemogenetic inhibition of cholinergic neurons in the left but not right MHb significantly decreased the expression of auditory cue-conditioned fear memory. Finally, we unilaterally injected Cre-expressing lentivirus into the MHb of GBR1-floxed mice to knockout GBRs. Deletion of GBRs in the left MHb attenuated fear expression, whereas its deletion in the right MHb enhanced fear expression, indicating a dichotomous role of MHb lateralization in emotion processing.Our study demonstrates synaptic functional asymmetry within the MHb-IPN pathway in mammals, highlighting its critical role in modulating fear-related behaviors, and offers new insights into the neural mechanisms underlying the regulation of emotions.