Bacteriophage infection leading to progeny production in a bacterial host requires timely expression of phage genes that is regulated by various phage- and bacteria-encoded factors. Mycobacteriophage D29, a lytic bacteriophage, is capable of infecting several mycobacterial species, including pathogenic Mycobacterium tuberculosis. Genomic characterization of D29 revealed two distinct promoters present at extreme ends of the genome that govern expression of phage genes. However, D29-derived transcriptional factors that regulate such expression remain largely unexplored. Here, we have characterized D29-encoded Gp36. We show that Gp36 binds to GC-rich direct repeats in sequence-specific manner. Gp36 makes weak homo-oligomer in vitro, with residues I25 and L35 being important for homo-oligomerization. We further show that Gp36 belongs to MerR family of transcriptional regulators, and represses expression of D29 genes; bacteriophage lacking gp36 shows higher expression of those early and late genes that are downstream to the Gp36 binding site in the genome. Such alteration of gene expression in mutated phage resulted in lower phage titer, although plaque size and host lysis timing remained unaltered. We thus present Gp36 as a transcriptional repressor of D29 with a regulatory role in modulating D29 gene expression, and envisage its engineering as a potential approach for developing phage therapeutics.