Kisspeptin, a neuropeptide encoded by KISS1/Kiss1 gene, and its cognate G protein-coupled receptor, GPR54 (Kiss-R) are critical in vertebrate reproductive control. In the zebrafish, two kisspeptin types are expressed in unique brain regions: Kiss1 neurons in the ventral habenula (vHb) and Kiss2 neurons in the hypothalamus. Recently we identified the role of habenular Kiss1 in serotonin (5-HT) system modulation with the suppression of alarm substance (AS)-evoked fear response in zebrafish. We also found that inactivation of the Kiss-R1-expressing neurons by injection of kisspeptin conjugated to saporin significantly decreased Kiss1-immunoreactivity in the vHb and MR as well as AS-evoked fear response. However, our morphological observation showed no direct contact between Kiss1 fibers and 5-HT neurons, suggesting an indirect mechanism of 5-HT modulation by Kiss1. Double-fluorescence labeling showed majority of Kiss1 neurons were glutamatergic and in the raphe region, Kiss1 fibers were noted in close association with glutamatergic and GABAergic cells, and very few fibers were closely associated with 5-HT cells. Hence, we postulated that majority of habenular Kiss1 neurons indirectly modulate the 5-HT system via glutamatergic and/or GABAergic interneurons. Some of the Kiss1 neurons could directly act on 5-HT cells, likely modulated via glutamatergic transmission as Kiss-R1 was not expressed in the 5-HT neurons. Therefore, our findings indicate that habenular Kiss1 neurons could inhibit fear response via the regulation of the 5-HT system through multiple aminergic transmission pathways.