Objective：To observe the effect on humoral immunity induced by deglycosylated middle hepatitis B virus surface antigen（MHBs） in BALB／c mice. Methods：By the use of gene modification using PCR，Asn encoding codon AAT／AAC were replaced by Gln encoding codon CAG， so the N-glycosylation sites Asn-X-Thr／Ser（NXS／T） were replaced by Gln-X-Thr／Ser（QXS／T）. On the base of a DNA vaccine（pSW3891／MHBs／Adr） encoding MHBs, three new N-linked deglycosylation mutations were engineered：dG1，deglycosylation of Asn 4，located on the preS2; dG23，deglycosylation of Asn 59 and Asn 146，located on the HBs and dG123, deglycosylation of Asn4, Asn59 and Asn146. In vitro expressions following transient transfetion in 293T cell line were observed. Also the MHBs antibody titers were studied in BALB／c mice by different deglycosylated MHBs DNA vaccine. Results：HBsAg could be detected both in the supernatant and the cell lysate after transient transfection in 293T cell line with adr and dG23 DNA vaccine. It also could be detected in the cell lysate but not in the supernatant with dG1 and dG123. Mice immunized with adr、dG1 and dG23 showed strong anti-HBs response（ending point>1：102400）, but not with dG123（ending point<1：200）. Conclusion：The MHBs secretion in 293T cells was significantly influenced by the deglycosylation of Asn 4 in MHBs， but the antibody level induced by deglycosylated vaccine in the mice was not affected. The MHBs secretion and the antibody titers were slightly influenced by the deglycosylation of both Asn59 and Asn146，but they were completely impacted by the deglycosylation of Asn4，Asn59 and Asn146 simutaneously.