The fertility of fish is a key factor in fish breeding. RNA-seq is widely used in high-throughput sequencing and provides a rapid method to examine the molecular mechanisms underlying a biological process. To probe fertility-related molecular mechanisms, we obtained testis transcriptomes from diploid and triploid cyprinid fish and tested for differentially expressed genes (DEGs) in the testis. A total of 6730 transcripts were differentially expressed between the triploid and diploid fish. In these transcripts, 2428 transcripts showed reduced expression and 4302 transcripts were overexpressed in triploid fish compared to the diploid fish. Functional analyses revealed that partial genes related to reproductive, developmental, and locomotion processes, and the axoneme, were differentially expressed in triploid fish relative to diploid fish. Pathway analysis indicated that variations in the gene expression levels of the “ubiquinone and other terpenoid-quinone biosynthesis pathway” and the “apoptotic pathway” played a central role in the sterility of triploid male fish. A series of genes (DNAHs, DNAL1, IFTs, and DNAAF1) associated with sperm flagellar assembly and motility, and testis-specific candidate markers (Tcte1, Tekt1, Tekt4, Spag17, Spag5, Spag9a, Spag1b, and Spef2), had low expression levels in the testis of triploid fish. We validated these DEGs in triploid fish using quantitative PCR to quantify expression of eight representative genes. Furthermore, 276 putative transcription factors, 6 chromatin remodeling factors, and 35 transcription cofactors exhibited differential expression in triploid compared to diploid fish. This study provides insight into the regulatory mechanisms causing sterility in male triploid fish.