Functionally uncharacterized gene Rv3627c is predicted to encode a carboxypeptidase in the pathogen of Mycobacterium tuberculosis (M. tuberculosis), which remains a major threat to human health. Here, we sought to reveal the function of Rv3627c and to elucidate its effects on mycobacterial growth. Rv3627c was purified from E. coli using Ni2+-NTA affinity chromatography, and its identity was confirmed with a monoclonal anti-polyhistidine antibody. An enzyme activity assay involving a D-amino acid oxidase-peroxidase coupled colorimetric reaction and high-performance thin layer chromatography was performed. A pull-down assay and MS-MS were also employed to identify putative interaction partners of Rv3627c. Scanning electron microscopy and transmission electron microscopy were performed to observe any morphological alterations to Mycobacterium smegmatis (M. smegmatis). We successfully obtained soluble expressed Rv3627c and identified it as carboxypeptidase using prepared peptidoglycan. Four proteins were identified as potential interaction partners with Rv3627c based on results obtained from both a pull-down assay and MS/MS analysis. Rv3627c over-expression induced M. smegmatis cells to become elongated, and promoted the formation of increased numbers of Z-rings. Rv3627c, a novel carboxypeptidase in M. tuberculosis identified in this study, exerts important effects on mycobacterial cell morphology and cell division. This functional information provides a promising insight into anti-mycobacterial target designs.