Background:Rousettus amplexicaudatus is one of the old world fruit bats, commonly known in Javanese word as codot belongs to Order Chiroptera and suborder Megachiroptera. R. amplexicaudatus possessed an outstanding visual ability, which helps in the process of searching for food. Those activities process and save within the brain involving the learning and memory activities. Learning and memory activities are centered around the hippocampus with the help of serotonergic nerve.Aim:This study conducted to identify the morphology and distribution of serotonergic nerve (5-hydroxytryptamine) in the hippocampus of R. amplexicaudatus, which related to the function of serotonin within the learning and memory process.Materials and Methods:Five R. amplexicaudatus bats were brought from Gunung Kidul cave, Yogyakarta, Java Island, Indonesia. The bats were anesthetized using ketamine and xylazine. R. amplexicaudatus then perfused intracardially. The brain samples were collected processed into paraffin block, and a further cut in 8 µm thickness serially. The tissue slides were stained using hematoxylin-eosin, cresyl echt violet, and immunohistochemistry with rabbit’s anti-serotonin (1:200). The results observed and analyzed quantitatively and qualitatively using image J (https://imagej.nih.gov/ij/) (Bethesda, Maryland, USA) and GraphPad Prism 7 (La Jolla, CA, USA).Results:The hippocampus of R. amplexicaudatus composed of the dentate gyrus (DG), hippocampus proper (cornu ammonis 3 [CA3], and CA1 as the main area for learning memory), and subiculum. On the DG serotonin immunoreactive cells found within the granular layer (132±35.03 cells/mm2), polymorphic stratum (86.33±11.23 cells/mm2), and molecular layer (93±1 cells/mm2). Meanwhile, on CA3 area, the immunoreactive cells for serotonin found in each stratum. The number of immunoreactive cells on each stratum from highest to the lowest are stratum pyramidal 123.33±15.88 cell/mm2, stratum molecular 63±13.11 cell/mm2, stratum lucidum 62.67±8.08 cell/mm2, stratum radiatum 55.33±510.21 cell/mm2, stratum oriens 48±3.46 cell/mm2, and stratum alveus 28.67±2,52 cell/mm2. In addition, in CA1 also hampers the immunoreactive cells in the pyramidal stratum, molecular, lucidum, oriens, radiatum, and alveus layer, respectively, of each 91±27.40 cell/mm2, 60.33±20.65 cell/mm2, 53.67±4.51 cell/mm244.33±10.40 cell/mm2, 41.33±5.51 cell/mm2, and 27±4 cell/mm2.Conclusion:Taking together the distribution of serotonin-immunoreactive cells in the hippocampus of R. amplexicaudatus mostly found on CA3 followed by CA 1 and DG.