Human cytomegalovirus glycoprotein complex Ⅱ(gCⅡ) consists of two glycoproteins, gM and gN. Although gCⅡ specific IgG purified from HCMV positive patient sera can neutralize HCMV, there has been no report on the generation of virus-neutralizing antibodies by immunizing with one epitope of gM. The epitope, termed MAD, was screened from random phage peptide library by subtractive strategy. The peptide sequence of MAD was highly homologous with 32～38 amino acids of HCMV gM. Mice immunized with MAD coupled with keyhole limpet hemocyanin (KLH) could produce specific antibodies against MAD, and the antibodies obtained could bind not only native HCMV particles, but also the recombinant gM30～78 peptide. ELISA analysis results showed that MAD could specifically bind HCMV-positive human serum samples. Virus-neutralizing assay results demonstrated that the antibodies against MAD could inhibit HCMV strain AD169 entering the human embryonic lung cells. The results suggested that MAD could be used as a new potential protective antigen in the development of HCMV vaccine.