AbstractSperm motility is the main index used to assess the quality of bull semen. It may also be used to evaluate the fertility potential of bulls. Protein-coding mRNA and long noncoding RNA (lncRNA) participate in the regulation of spermatogenesis. Here, we employed strand-specific RNA sequencing to profile the semen transcriptome (mRNA and lncRNA) of six paired full-sibling Holstein bulls with divergent sperm motility and to determine the functions of mRNA and lncRNA in sperm motility. Among 20,875 protein-encoding genes detected in semen, 19 were differentially expressed between the high sperm motility group (H: H1, H2, and H3) and low sperm motility group (L: L1, L2, and L3). Of the 11,561 lncRNAs identified in sperm, 2,517 were differentially expressed between the H and L groups. We found that TCONS_00041733 lncRNA targets the node gene EFNA1 (ephrin A1), involved in male reproductive physiology. Our study provides a global mRNA and lncRNA transcriptome of bull semen, as well as novel insights into the regulation of neighboring protein coding by lncRNAs and the influence of mRNAs on sperm motility.