Marek's disease (MD), caused by Marek's disease virus (MDV), is a lymphoproliferative neoplastic disease of chickens and is characterized by MD lymphoma in multiple visceral organs of chicken. It causes great damage to poultry health. Recently, miRNA has been reported to be involved in Marek's disease lymphomagenesis. Our previous study showed that gga-miR-181a was downregulated in MDV-induced lymphoma, and its target gene, v-myb myeloblastosis viral oncogene homolog-like 1 (MYBL1), was predicted. In this study, the interaction between gga-miR-181a and MYBL1 was further verified by detecting protein expression levels of MYBL1 after transfecting miR-181a mimic into MD lymphoma cell line, MSB1. The result showed that protein level of MYBL1 was lower in gga-miR-181a mimic transfecting group than that in the negative control group at 96 h post transfection, which indicated that MYBL1 was a target gene of gga-miR-181a. Additionally, we found that the expression of MYBL1 was higher in MDV-infected samples than that in non-infected controls, which agreed with the proposition that miRNA showed a negatively correlated expression pattern with its target gene. We observed the inhibitory effect of gga-miR-181a on MSB1 cell proliferation. Collectively, the aberrant expression of gga-miR-181a and MYBL1 in MD lymphoma suggested that they might be involved in MD tumor transformation and played important roles.