SUMMARY Euryhaline tilapia Oreochromis mossambicus acclimates to the external spawning environment by modulating its mechanism for regulating sperm motility. Adaptation of sperm was performed by acclimating fish in various environments. In this paper, regulatory mechanisms of freshwater-acclimated tilapia were studied in detail. Tilapia sperm motility was vigorous in hypotonic conditions and decreased with increasing osmolality. Sperm motility was reduced in hypotonic conditions when extracellular Ca 2+ was chelated; however, extracellular Ca 2+ was not a major factor for motility activation since sperm were motile even when extracellular Ca 2+ levels were nominally depleted by EGTA. The Ca 2+ indicator, fluo 3, showed that intracellular [Ca 2+ ] increased on motility activation independently of extracellular [Ca 2+ ],accompanied by swelling of the sperm neck region called the sleeve structure. Intracellular [Ca 2+ ] was not increased under hypertonic conditions,in which sperm were immotile, even on addition of extracellular Ca 2+ . It is possible that Ca 2+ is stored in the neck region. Demembranated sperm were reactivated in the presence of Ca 2+ , but cAMP failed to reactivate the motility. Furthermore, we detected phosphorylation and dephosphorylation of three proteins at serine and threonine residues on motility activation. It is likely that hypotonic shock causes an increase in intracellular [Ca 2+ ] that activates motility activation via phosphorylation of some flagellar proteins.