Sperm motility is a crucial trait in chicken production, and the epididymis is an essential organ in the reproductive system. Currently, the molecular mechanisms underlying sperm motility in the epididymis are unclear. In this study, 8 cDNA libraries and eight miRNA libraries were constructed from roosters (4 chickens per group) with diverse sperm motility. After a comparative analysis of epididymal transcriptomes, we detected 84 differentially expressed genes (
DEG
s) using the edgeR package. Integrated interpretation of DEGs indicated that
MMP9, SLN, WT1, PLIN1
, and
LRRIQ1
are the most promising candidate genes affecting sperm motility in the epididymis of roosters. MiR-146a, mir-135b, and mir-205 could play important regulatory roles in sperm maturation, capacitation, and motility. Additionally, a comprehensive analysis of the mRNA and miRNAs transcriptomes in silico identified a promising gene-miRNA pair miR-135b-HPS5, which may be a vital regulator of sperm motility in the epididymis. Our findings provide novel integrated information of miRNAs and genes that shed light on the regulatory mechanisms of fertility in roosters.