Mycobacterium tuberculosis H37Ra (Mtb-Ra) ORF MRA_1916 is annotated as a D-amino acid oxidase (DAO). These enzymes perform conversion of d-amino acids to corresponding imino acids followed by conversion into α-keto-acids. In the present study Mtb-Ra recombinants with DAO knockout (KO) and knockout complemented with DAO over-expressing plasmid (KOC) were constructed. The growth studies showed loss of growth of KO in medium containing glycerol as a primary carbon source. Substituting glycerol with acetate or with FBS addition, restored the growth. Growth was also restored in complemented strain (KOC). KO showed increased permeability to hydrophilic dye EtBr and reduced biofilm formation. Also, its survival in macrophages was low. Phagosome maturation studies suggested enhanced colocalization of KO, compared to WT, with lysosomal marker cathepsin D. Also, an increased intensity of Rab5 and iNOS was observed in macrophages infected with KO, compared to WT and KOC. The in vivo survival studies showed no increase in CFU of KO. This is the first study to show functional relevance of DAO encoded by MRA_1916 for Mtb-Ra growth on glycerol, its permeability and biofilm formation. Also, this study clearly demonstrates that DAO deletion leads to Mtb-Ra failing to grow in macrophages and in mice.