This study, for the first time in fish, compared the transcriptome of fresh and frozen-thawed sperm, and would help to better understand the effect of cryopreservation on fish sperm and then better preserve the aquatic germplasm resources. Here, we employed high-throughput sequencing technology to obtain the transcriptome of yellow catfish from fresh sperm, cryopreserved sperm with and without cryoprotectant. When cryoprotectant (Me2SO) was excluded, down-regulated genes were significantly enriched into calcium ion binding, cytoskeletal protein binding, microfilament motor activity, calmodulin binding and carnitine O-acyltransferase activity, which affected Ca2+ regulation, cellular morphology, motility and metabolism. Moreover, heat shock proteins and genes associated with regulation of cholesterol, HCO3- and protein tyrosine phosphorylation (PTP) were down-regulated, and thus would impair ability against stress, membrane rigidity, pH regulation and signal transduction of cryopreserved sperm. After Me2SO was added, the amounts of DEGs decreased significantly and down-regulation of genes were found mainly in cytoskeleton and heat shock proteins, thereby suggesting that Me2SO effectively reduced the impact caused by low temperature on gene expression. Whether adding Me2SO or not, the up-regulated genes were mainly found in ribosomal proteins genes. However, when Me2SO was added, over-expression of some genes might contribute to maintain normal function of cryopreserved sperm.