Serotonin (5-HT), a neurotransmitter, is involved in the regulation of a wide variety of physiological functions, such as sensory, motor, memory, mood, and reproduction. The 5-HT system regulates gonadotropin-releasing hormone (GnRH) and gonadotropin release. Recently, kisspeptin acting upstream to GnRH has been identified as an important regulator of reproduction, however the functional association between 5-HT with kisspeptin systems remains unknown. In the mature male zebrafish, there was no association between 5-HT fibers with hypothalamic kisspeptin (Kiss2) cell bodies or between 5-HT fibers and Kiss2 fibers in the pituitary, while we found close associations between 5-HT fibers and Kiss2 fibers in the preoptic area (POA), ventral (HV) and caudal hypothalamic (HC) regions, suggesting serotonergic action on Kiss2 via fiber-fiber associations. Two weeks treatment with citalopram, a selective serotonin reuptake inhibitor (4, 40 and 100 μg/L) significantly decreased mRNA levels of kiss2 in the brain and gonadotropins beta-subunits (lhb and fshb) in the pituitary. In addition, the intensity of Kiss2 and 5-HT fibers and the number of their close associations were significantly decreased in the POA, HV and HC. However, there was no effect of 2 weeks of citalopram treatment on the profiles of different stages of spermatogenesis and matured sperm count. One month of citalopram treatment (40and100μg/L) significantly decreased mRNA levels of kiss2 and gonadotropins, as well as sperm production.These results suggest that the serotonergic action on Kiss2 system may play an important role in the control of reproductionin the male zebrafish.