Abstract This study presented information about the semen evaluation of captive male hawksbill sea turtles, Eretmochelys imbricata, based on an extended 15-mo study using the electro-ejaculation technique. In particular, we demonstrated that hawksbill sperm, which is underactive just after ejaculation, was activated by the presence of urine. The findings are useful for developing optimal semen collection techniques for future artificial insemination programs of hawksbill turtles.