The aims of this study were to determine the relation of glucose transporter 3 (GLUT3) expression on the spermatozoa membrane with the quality of semen in Asian elephants. Fresh semen samples were collected from 10 Asian elephants and classified according to the percentage of motile sperm into Group 1 (≤ 20%; n = 4 males), Group 2 (>20% to 60%; n = 3 males), and Group 3 (>60%; n = 3 males). The immunocytochemistry results showed the presence of GLUT3 at the principal and end piece of the sperm tail. GLUT3 expressions in each group were significantly different (P < 0.05). The group with good sperm motility (Group 3) significantly (P < 0.05, R = 0.960) expressed higher GLUT3 (99.40 ± 0.69%) than Group 1 (21.46 ± 10.25%) and Group 2 (84.37 ± 8.70%). Moreover, the percentage of live sperm of this group was also significantly highest (P < 0.001, R = 0.938) at 82.00 ± 4.00% compared to Group 1 (20.75 ± 3.30%) and Group 2 (53.00 ± 8.02%). Therefore, the motility of Asian elephant spermatozoa and percentage of live sperm may be influenced by GLUT3 expression, and GLUT3 expression may be involved in energy production via the glycolytic pathway.