In this study, effects of amino acids, glutamine and proline, as supplements of freezing extender were studied during cryopreservation of stallion spermatozoa. The CASA and biochemical assays were performed following thawing of sperm that had been frozen in basal medium as control (E1) and basal medium containing 3-mM (E2), 30-mM (E3), and 60-mM proline (E4); 5-mM (E5) and 50-mM glutamine (E6); and 3-mM proline plus 5-mM glutamine (E7). E2 increased progressive motility (P < .001), motile spermatozoa (P > .05), values of curvilinear velocity, average path velocity, mean angular displacement (MAD), lateral head displacement, beat cross-frequency, and straightness (STR) compared to control extender. E3 led to a less motile and progressive motile sperm. E3, E4, and E6 were found to provide for lower mean linearity (LIN), wobble, and STR values than control group. E5 increased LIN value and decreased MAD value significantly (P < .05). CASA parameters of E7 were not different with control. The levels of malondialdehyde did not change. The glutathione peroxidase activity remained unchanged with E2, E3, E4, and E5 but decreased in the E6 (1,933 ± 210.1) and E7 (1,976.5 ± 5.8), although the change was not significant (P > .05). E3 (2,080 ± 288) and E5 (1,553.3 ± 155.8) significantly increased catalase activity (P < .05), and superoxide dismutase percent inhibition with E7 (38.46 ± 28.2) was significantly higher than other groups (P < .05). The higher motility of stallion spermatozoa in diluent containing 3-mM proline may allow for the more efficient use of frozen-thawed stallion semen for insemination and could contribute to the improvement of semen cryopreservation in the world horse industry.