Sperm motility is a critical determinant of male fertility in vertebrates including fish species. Chloroquine, an autophagy inhibitor, has been reported to own debated influence on sperm quality of mammalians. The objectives of this study were to determine the effect of chloroquine on sperm motility and fertilizing efficiency in yellow catfish, an important freshwater fish species in China. Gradient doses of chloroquine were intraperitoneally injected into yellow catfish that resulted in an increasing expression level of LC3-II and inhibition of autophagy. Subsequently, various sperm parameters were assessed with the computer-assisted sperm analysis (CASA) system. Chloroquine injection significantly increased straight line velocity (VSL), curvilinear velocity (VCL) and average path velocity (VAP) of sperm compared with the control. Consequently, 1 mu M chloroquine/g body weight was revealed to be an optimal dose for in vivo treatment, which led to elevation of fertility rate, whereas no effect on the hatching rate. The comparative transcriptome analysis between chloroquine-treated and control testes were conducted in order to figure out the possible molecular mechanism of chloroquine to regulate sperm motility. Interestingly, Gene Ontology (GO) analysis indicated that chloroquine treatment not only inhibited autophagy pathway but also significantly reduced toll-like receptor signaling pathway, suggesting a possible trade-off between male reproduction and immunity. KEGG analysis showed up-regulation of many pathways including PI3K-AKT signaling pathway which has been reported to be correlated with spermatogenesis and sperm maturation in yellow catfish. Our data reveals a potential trade-off between reproductive traits and immune function, and suggests that the application of chloroquine could improve sperm quality and fertilization efficiency in broodstock fish. Statement of relevance: Our research demonstrated that an optimal dose of chloroquine could efficiently inhibit autophagy and improve sperm motility and fertilization efficiency. The comparative transcriptome analysis between chloroquine-treated and control testes were conducted in order to figure out the possible molecular mechanism of chloroquine to regulate sperm mobility. (C) 2016 Elsevier B.V. All rights reserved.