In this study, sperm characteristics of Black Sea trout (Salmo trutta labrax) and effects of ions, their concentrations and osmolality of the solution on sperm activation were evaluated. Two different activation solutions with ionic and non-ionic osmolality levels were investigated. After the definition of conditions where osmolality played a role, activation solutions containing calcium ion (Ca2+) in different molarity levels were tested under single and combined solutions to determine their relations (Sperm motility percentage was determined by using computer-aided sperm analysis system (CASA) for each activation solution). It was found that Black Sea trout sperm motility is initiated with iso-osmolality and activation solution with non-ionic osmolality had no effect on motility whereas ionic osmolality was effective on motility. The best solution determined in this study for the Black Sea trout is proved to have the same osmolality as the seminal plasma of the handled fish prepared with NaCl containing 2–32 mM CaCl2.