Epidermal growth factor (EGF)-like (EGFL) domain, a common structural module in numerous secreted or transmembrane proteins, is generally involved in protein-protein interactions. To date, several EGFL proteins have been identified and characterized, but little is known about EGFL domain 8 (EGFL8). The present study reported the molecular characterization and expression analysis of EGFL8 in mice. Mouse EGFL8 amplified using a reverse transcription-polymerase chain reaction approach was sequenced and characterized. Mouse EGFL8 encodes a protein of 293 amino acids with two EGFL domains, an Emilin-like domain and a Ca(2+)-binding EGFL domain, which has a molecular mass of 32 kDa. The coding sequence has a high degree of amino acid sequence identity across species, and the EGFL domain has been highly conserved in various species during evolutionary radiation. A phylogenetic tree calculated using the neighbor-joining method revealed that EGFL8 and EGFL7 are more closely associated with each other than either is to EGFL3, and they cluster with EGFL6. It was found that mouse EGFL8 protein was highly expressed in diverse mouse tissue types, including the thymus, lymph nodes, testis, ovaries, epididymis, ductus deferens, ileum, colon, stomach, esophagus, lung, uterus, urinary bladder, skin, spleen, adrenal glands and penis. These results are of great use in understanding the biological roles of mouse EGFL8 for further study.