Until recently, the role of the proteolytic system involving serine proteases in follicle rupture during ovulation in mammalian species has been a subject of controversy. We undertook the present study to examine whether proteases play a role in follicle rupture using the teleost medaka (Oryzias latipes) model. Various serine protease inhibitors, including a specific plasmin inhibitor, drastically reduced the rate of ovulation, as assessed by an in vitro ovulation assay, which was established for the fish. Biochemical, molecular biological, and immunological analyses demonstrated that plasminogen/plasmin was present in large follicles destined to ovulate. The active protease, plasmin, was detected in follicles approximately 3–7 h before the expected time of ovulation. Specific antibodies against the medaka plasmin light chain suppressed the ovulation rate of the follicles when antibodies were added to the medium during the period in which active plasmin was generated. This finding was an indication that a plasmin-like protease similar if not identical to plasmin plays a role in follicle rupture during ovulation in the medaka. Our data also indicate that this serine protease participates in the rupture for only a few hours prior to the activation of matrix metalloproteinase (Mmp)-mediated hydrolysis at ovulation. Based on our previous and current data, we propose a follicle rupture model involving two different proteolytic enzyme systems, serine protease and Mmp, in medaka ovulation. The current study is the first to provide evidence of the indispensable role of plasmin or a plasmin-like protease in the ovulation of a nonmammalian vertebrate species.