The protective efficacy of a non-replicating Adenovirus serotype 5, expressing the immunogenic envelope glycoprotein B (Ad5-gB) of Mareks disease virus, was investigated in a vaccine-challenge model for Mareks disease in experimental chickens. In ovo vaccination with Ad5-gB, with or without a second vaccination posthatch, was compared with pCVI988 (a clone of the gold-standard CVI988 Mareks disease vaccine). In ovo vaccination with Ad5-gB, without the second vaccination, gave a protective index of 37.5%, but did not reduce replication, shedding or transmission of virulent virus. In ovo vaccination followed by a post-hatch vaccination with Ad5-gB, was as protective as pCVI988 against mortality and Mareks disease lesions (100% protection) and, like pCVI988, efficiently reduced the level of virulent virus in the blood of chickens. However, although this double-dose Ad5-gB vaccination delayed the onset of shedding of virulent virus, it did not inhibit shedding and was less effective than pCVI988 in reducing shedding and transmission of virulent virus. Further optimisation of Ad5-gB dose, administration route and time of vaccination could lead to trials as a potential vectored vaccine candidate for Mareks disease, with a number of advantages over the current live cell-associated vaccines: no requirement for maintenance of a cold chain during vaccine preparation and administration, no horizontal spread, reduced selection pressure for highly virulent virus, and no possibility of reversion to virulence.