Infectious diseases are the primary cause of neonatal morbidity and mortality in people, resulting in millions of deaths every year. Infection of the newborn with some of the pathogens involved, such as herpes simplex virus (HSV), human immunodeficiency virus (HIV), hepatitis B virus (HBV), human cytomegalovirus (HCMV) or group B Streptococcus sp. (GBS), usually occurs at the end of pregnancy, during birth or by breast feeding. Therefore, active immunization of the fetus might represent an effective approach to reduce the high risk of neonatal diseases. We recently showed that DNA immunization in utero within the third trimester of gestation induced strong humoral and cell-mediated immune responses in immunized fetal lambs. Here, we demonstrate that fetal immunization was safe and did not affect fetal gestation, neonatal viability, or significantly alter blood leukocyte populations. In utero immunization resulted in the induction of protective mucosal immunity and immune memory in the newborn lamb. Furthermore, there was no evidence that in utero DNA immunization induced immune tolerance. Our results also indicate that the uptake and expression of the plasmid DNA already occurred within the epithelium of the oral cavity. This correlates with our previous findings that local immune responses were found exclusively in the retropharyngeal lymph nodes draining the oral cavity.