Osmolality and composition of the activating solution on motility of fresh and frozen Prochilodus lineatus sperm were evaluated. Sperm was triggered in 11 solutions prepared with reverse osmosis (RO) water (∼0mOsmkg−1), and glucose or NaCl adjusted to 50, 100, 150, 200 and 250mOsmkg−1. Sperm motility rate and velocities (curvilinear=VCL, among others) were evaluated in fresh sperm at 10, 30 and 50s post-activation (spa), and in frozen sperm at 10 spa only. Sperm was frozen under a standardized methodology for this species. Fresh sperm motility was higher in samples triggered in RO (91%), in glucose at all osmolalities (90–92%) and in 50–150mOsmkg−1 NaCl (88–91%) than that in 200–250mOsmkg−1 NaCl (74–80%). Motility decreased (P<0.05) as a function of time after activation in samples activated in RO and in NaCl but not in glucose. Samples activated in 100–250mOsmkg−1 glucose yielded motility above 80%, at 50 spa. Curvilinear velocity was higher (P<0.05) in glucose-activated samples (322–357μms−1) compared to that activated in NaCl (192–283μms−1) and in RO (298μms−1). Frozen sperm motility and velocities were similar when triggered in RO, glucose or NaCl and were higher at 0–150 mOsm kg−1 (69–78% motility; 163–208μms−1 VCL) than at 200–250mOsmkg−1 (34–59% motility; 127–168μms−1 VCL). High sperm motility with fast velocity for a long period is achieved at 100–150mOsmkg−1, in glucose solution for fresh sperm and in glucose or NaCl for frozen sperm.