The effect of hepatitis B immunoglobulin (HBIG) on hepatitis B virus (HBV) DNA load and its protective mechanism are not well understood. Twenty-eight hepatitis B surface antigen (HBsAg)-positive pregnant women and their newborns were assigned to an experimental (n = 12) or control group (n = 16) according to whether they received HBIG during pregnancy. HBV DNA load and markers titer of the mothers and newborns were tested. These markers and HBV DNA load in mothers of the experimental group did not fluctuate significantly and were comparable to the control. In the experimental group, there was a positive correlation between mothers and their newborns with regard to hepatitis B surface antibody titer. Immunohistochemical staining of placenta sections showed that HBsAg-positive areas mainly included trophoblastic cells and villous mesenchymal cells without HBIG colocalization, whereas HBIG-positive areas principally included villous capillary endothelial cells and villous mesenchymal cells. Additionally, compared with the control group, the positive rate and mean density of HBIG in the experimental group were remarkably higher. HBIG deposition was seen in Hofbauer cells. Thus, rather than influencing virus replication, HBIG forms an immune barrier between the mother and fetus to prevent HBV transmission.