Amino acid supplementation is an efficient and effective strategy to increase sperm quality. In our research, a comparative study was conducted to screen free amino acids to improve sperm motility, and we found that leucine was the most efficient one. Leucine treatment increases sperm motility depending on the activation of PI3K/Akt signaling pathway, while the chemical inhibitor of PI3K/Akt signal could reduce the amount of pAkt activated by leucine treatment. Moreover, leucine treatment improved the expression of P62 and LC3-II, substantially suppressed the autophagy process in zebrafish testis. In vitro studies showed that leucine could reduce the fusion of autophagosome and lysosome that was indicated by the co-localization of EGFP-LC3 and lysosome marker. Two chemical modulators of autophagy, such as LY294002 (the inhibitor of PI3K/Akt signal) and chloroquine were administered to investigate the process of autophagy on zebrafish sperm motility. LY294002 inhibited autophagosome formation to reduced sperm motility, while chloroquine inhibited the fusion of autophagosome and lysosome to improve sperm motility. Our data suggest that short-term treatment with leucine could increase zebrafish sperm motility by affecting the autophagy and inhibiting the fusion of autophagosome and lysosomes, depending on the activation of PI3K/Akt signaling pathway.