Glycoprotein B (gB) has emerged as a subunit-vaccine candidate for congenital cytomegalovirus (CMV) infection, a major public health problem. The present study evaluated a cloned, recombinant gB vaccine in the guinea pig cytomegalovirus (GPCMV) model of congenital infection. Guinea pigs were immunized with gB, which was coadministered with either Freund's adjuvant or alum. All gB-immunized dams had enzyme-linked immunosorbent-assay and neutralizing-antibody responses, with significantly higher titers in the gB/Freund's group. Pregnant dams were challenged with GPCMV subcutaneously during the 3rd trimester. Maternal DNAemia on day 10 after infection trended lower in gB-immunized dams than in control animals, with significant reductions in the gB/Freund's group. Vaccination resulted in a highly significant reduction in pup mortality. For the gB-vaccine groups, pup mortality was significantly lower, and reduced rates of GPCMV transmission were noted, for dams immunized with gB and Freund's adjuvant, compared with dams immunized with gB and alum. These are the first data indicating that a recombinant gB vaccine protects against congenital CMV infection and disease.