Long intergenic non-coding RNAs (lincRNAs) are transcribed from non-coding DNA sequences. Studies have revealed that aberrant expressions of lincRNAs are associated with various types of cancers and neurological disorders. Marek's disease (MD) is a highly contagious T-cell lymphoid neoplasia of chicken induced by Marek's disease virus (MDV). In this study, we first identified and validated linc-GALMD3 highly expressed in MDV-infected CD4+ T cells by RNA-Seq and qRT-PCR. By RNA-Seq analysis in MDCC-MSB1 cells after loss of function of linc-GALMD3 by shRNA, we found that linc-GALMD3 could positively cis-regulate its downstream gga-miR-223 gene expression. In contrast, it could trans-regulate the 748 differentially expressed genes (FDR < 0.01) that were mainly enriched into mitochondrial structure and cell cycle processes using GO analysis. Of these, the most significantly expressed gene EPYC might cause iris lesion in MD. The other eight genes, NDUFA4, NDUFB6, NDUFV1, NDUFS8, SDHB, UQCRC1, UQCRC2, and COX7A2, actively participated in oxidative phosphorylation in mitochondrial dysfunction and cell death. Most importantly, we found that the MDV replication was repressed when linc-GALMD3 was knocked down in CEF cells. Our results suggested that linc-GALMD3 might be a critical regulator in chicken MD and could be used as a candidate-promising mark for MD prevention, diagnosis, and treatment.