Factors that inhibit and stimulate the initiation of sperm motility were determined for Manila clam (Ruditapes philippinarum), Pacific oyster (Crassostrea gigas), and Japanese scallop (Patinopecten yessoensis). Compared with artificial seawater (ASW), serotonin (5-hydroxytryptamine creatinine sulfate, 5-HT) could fully trigger sperm motility and increase sperm velocity and motility duration. Sperm motility was decreased in ASW at pH 6.5–7.0 and suppressed at pH 4.0. In Manila clam and Pacific oyster, 5-HT could overcome the inhibitory effects of acidic pH on sperm motility. In the presence of nigericin (a K+/H+ exchanger), sperm motility was only triggered at pH 8.3. Testicular fluid K+ concentrations were two- to fourfold higher than that in ASW. Sperm motility and velocity were decreased in ASW or 5-HT containing ≥40 mM K+ or ≥2.5 mM 4-aminopyridine, suggesting K+ efflux requirement to initiate motility. Sperm motility and velocity were reduced in ASW or 5-HT containing EGTA or W-7, suggesting that extracellular Ca2+ is required for Ca2+/calmodulin-dependent flagellar beating. Ca2+ influx occurs via Ca2+ channels because sperm motility and velocity were decreased in both ASW and 5-HT containing T-type and L-type Ca2+ channel blockers. 5-HT-dependent initiation of sperm motility was associated with intracellular Ca2+ rise, which was comparable to that seen in ASW but was not observed in the presence of EGTA or a Ca2+ channel blocker. Extracellular Na+ is also essential for sperm motility initiation via regulation of Na+/Ca2+ exchange. Overall, 5-HT-dependent initiation of sperm motility in marine bivalve mollusks is an osmolality-independent mechanism and regulated by extracellular pH, K+, Ca2+, and Na+.