Relaxin3 (RLN3) is involved in arousal, feeding, stress responses, cognition, osmoregulation and anxietyrelated behaviors in mammals.Although two rln3 paralogues have been characterized in teleosts, their functions still remain largely unknown.We identified two rln3 paralogous genes, represented as rln3a and dn3b, from Nile tilapia (Oreochromis niloticus) gonad.Rln3a was dominantly expressed in testis followed by brain, pituitary and kidney.Contrastingly, rln3b was most abundant in brain, even though low transcription was detected in pituitary and testis.Ontogenic analysis revealed that testicular rln3a expression increased from 30 days after hatching (dah) and surged dramatically from 150 dah.However, significant enhancement of rln3b expression was detected only from 150 dah.Similarly, flourescence in situ hybridization detected Leydig cells specific rln3a and rln3b expression from 90 and 150 dah, respectively.CRISPR/Cas9 based transcriptional silencing of rln3a gene repressed the transcription of germ and Leydig cell marker genes, but not Sertoli cell markers, and temporarily retarded the spermatogenesis at 90 dah.Interestingly, 150 dah onwards, spermatogenesis was gradually restored with the occurrence of spermatocytes.By 240 dah, rln3a deficient XY fish were able to produce fertile sperm, however, the amount of sperm and their fertilization rate was substantially reduced.Similar phenotype was also found in F1 heterozygotes at 90 dah.On the other hand, acute salinity stress significantly induced rln3b expression, but not rln3a, in the brain at both 8 and 20 ppt.Taken together, we provide further evidences that the two paralogous of rln3 in tilapia might be involved in distinct physiological pathway, especially, rln3a in spermatogenesis and dn3b in osmoregulation.