The VapBC system, which belongs to the type II toxin–antitoxin (TA) system, is the most abundant and widely studied system inMycobacterium tuberculosis. The VapB antitoxin suppresses the activity of the VapC toxin through a stable protein–protein complex. However, under environmental stress, the balance between toxin and antitoxin is disrupted, leading to the release of free toxin and bacteriostatic state. This study introduces the Rv0229c, a putative VapC51 toxin, and aims to provide a better understanding of its discovered function. The structure of the Rv0229c shows a typical PIN-domain protein, exhibiting an β1-α1-α2-β2-α3-α4-β3-α5-α6-β4-α7-β5 topology. The structure-based sequence alignment showed four electronegative residues in the active site of Rv0229c, which is composed of Asp8, Glu42, Asp95, and Asp113. By comparing the active site with existing VapC proteins, we have demonstrated the justification for naming it VapC51 at the molecular level. In an in vitro ribonuclease activity assay, Rv0229c showed ribonuclease activity dependent on the concentration of metal ions such as Mg2+and Mn2+. In addition, magnesium was found to have a greater effect on VapC51 activity than manganese. Through these structural and experimental studies, we provide evidence for the functional role of Rv0229c as a VapC51 toxin. Overall, this study aims to enhance our understanding of the VapBC system inM. tuberculosis.