Cryopreservation of fish sperm is a valuable method for restoration of endangered species as well as a technique for manipulation of reproduction for genetic improvement in fish. The objective of this study was to determine the effect of equilibration time, vapour temperature and exposure time on postthawed sperm motility characteristics in red tilapia. Semen was collected from matured male, diluted using TCAYE extender in French straws (0.25 ml) and stored in liquid nitrogen tank. This research involved a 3 x 4 x 4 factorial experiment consisting of 3 equilibration times (30, 45 or 60 minutes), 4 vapour temperatures (-70, -80, -90 or -100°C) and 4 exposure times (5, 7, 9 or 10 minutes). Sperm movement and velocity distribution after frozen-thawed were evaluated using Automated Semen Analyzer (IVOS, Hamilton-Thorne). The highest percent motility was obtained significantly (P ≤0.05) when red tilapia fish sperm were equilibrated for 60 minutes (63.2±1.9%), vapourized at -80°C (61.2±2.1%) and exposed for 10 minutes (57.6±2.0%). The results from this study on red tilapia fish sperm diluted with TCAYE extender suggested that the optimal percent motilities of sperm could be obtained from combination of 60 minutes equilibration time, -80°C of vapour temperature and 10 minutes of exposure time.