Herpesvirus envelope glycoproteins play important roles in mediating infection initiation and also represent major immunogens. We recently showed that a pseudorabies virus (PrV) mutant lacking the essential glycoprotein gD (gp50), after phenotypic complementation by propagation on genetically engineered PrV gD-expressing cell lines was able to infect primary target cells and spread exclusively by means of direct cell-to-cell transmission. Virions released from non-complementing cells that lacked gD were not infectious because of a defect in penetration and so free infectious virions did not arise after infection of animals by phenotypically complemented gD-negative PrV. This formed the basis for the development of novel non-spreading live herpes-virus vaccines. However, the gD-negative PrV mutant still retained a residual level of virulence, which prevented its use as vaccine, and the need to propagate the gD-negative PrV mutant on trans-complementing cell lines resulted in the appearance of wild-type revertants, rescued by the resident gene in the cell line. To overcome these problems we isolated a PrV mutant designated PrV(376) that, in addition to gD, also lacked the non-essential glycoproteins gG, gI and gE. PrV(376), because of the lack of gD, was also dependent on gD-expressing cells for productive replication. Non-complementing cells infected by phenotypically gD-complemented PrV(376) produced non-infectious particles lacking glycoproteins gD and gE as shown by immunoelectron microscopy. Owing to the absence of any homologous sequences between the viral genome and the viral genes resident in the complementing cell line, rescue by homologous recombination was impossible. In cell culture, plaques of PrV(376) were significantly smaller than those of either wild-type, or gD- or gE-deleted mutants, indicating an additive or synergistic effect of the combined deletion on viral cell-to-cell spread capability. Intranasal or intramuscular infection of pigs with phenotypically gD-complemented PrV(376) showed a complete attenuation of viral virulence, with an expected lack of shedding of infectious virus. The PrV(376)-vaccinated pigs exhibited a significant level of protection against challenge infection, measured by survival and weight loss. In summary, PrV(376) represents a novel type of herpesvirus vaccine that combines innocuity, efficacy and biological safety.