Technical Abstract: Marek’s disease is a devastating neoplastic disease of chickens caused by gallid herpesvirus 2 or Marek’s disease virus (MDV), which is characterized by massive visceral tumors, immune suppression, neurologic syndromes, and peracute deaths. It has been reported that MDV down-regulates surface expression of major histocompatibility complex (MHC) class I molecules, although the mechanism has remained elusive. Close relatives of MDV have been shown to down-regulate MHC class I expression through small viral proteins, such as the infected cell protein (ICP) 47 of herpes simplex virus 1 or the unique long (UL) 49.5 proteins of a number of varicelloviruses, including bovine herpesvirus 1 and pseudorabies virus. MDV does not encode ICP47, but harbors a UL49.5 homologue. Therefore, we tested the contribution of MDV UL49.5 protein (pUL49.5) to down-regulation of MHC class I expression. Using in vitro assays, we conclusively showed that MDV pUL49.5 down-regulates MHC class I directly in the absence of other viral gene products. Additionally, we identified the cytoplasmic tail of MDV pUL49.5 as essential for this function. When viruses lacking the cytoplasmic tail of pUL49.5 were tested in vivo, no differences were seen in highly susceptible chickens of the B19B19 MHC class I haplotype with respect to the development of MD, and horizontal transmission compared to wild-type and revertant viruses. In contrast, there was a reduction in pathogenic potential of the deletion viruses in B21B21 chickens that are more resistant to MDV infection. We concluded that the effect of an inability to down-regulate MHC class I on MDV virulence is small but dependent on the MHC class I haplotype.