Carboxylesterases (CEs) represent a multigene family of serine-dependent enzymes. Male-dependent CEs are over-expressed in the male reproductive tract of different animal species (bivalve mollusks, fruit-flies, and mammals). Here, a novel rat epididymis-specific gene named Ces7 was cloned and characterized. It was a novel member of CE family, which was mainly expressed and secreted to the lumens of the corpus and cauda epididymis. CES7 protein was highly glycosylated as other mammalian CEs. Furthermore, Ces7 increased with age growth until sex maturation and then maintained at high level. CES7 might be one of the major CEs in male reproductive tract and contribute to the sperm fertilization.