This study aims to investigate the function of Rv3425 in Mycobacterium tuberculosis (M. tuberculosis) by screening its interacting proteins in bacillus Calmette­Guérin (BCG) via co-­immunoprecipitation coupled with mass spectrometry. Regions coding for Rv3425 were amplified by polymerase chain reaction (PCR) and cloned into the pMV261 vector. The recombinant vector was transformed into BCG, and the expression of the fusion protein was verified by Western blotting analysis. The Rv3425 protein complex formed in BCG (rBCG) were obtained by co-­immunoprecipitation combined with anti­-His specific antibody. Mass spectrometry was used to identify protein components in the complex; the function of each protein was obtained from the National Center for Biotechnology Information (NCBI). The candidate proteins interacting with Rv3425 were verified by glutathione S­-transferase (GST) pulldown assay. Ten proteins interacted with Rv3425 were obtained. Rv3425 could coordinate with a variety of proteins implicated in intermediary metabolism and respiration, cell wall and cell processes as well as latent infections. The results also demonstrated that Rv3614c protein implicated in ESX­1 secretion system and possible pathogenic mechanism could bind to Rv3425 in vitro.