Mycobacterium massiliense is an emerging pathogen and very similar to Mycobacterium abscessus of rapidly growing mycobacteria in the phenotype and genotype. Pathogenic bacteria secrete a diversity of factors into extracellular medium which contribute to the bacterial pathogenicity. In the present study, we performed the comparative proteome analysis of culture filtrate proteins from a clinical isolate of M. massiliense and M. abscessus strains using two-dimensional gel electrophoresis and liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS). Interestingly, 9 proteins of M. massiliense were distinctly expressed from those of M. abscessus. Bioinformatic analysis of the identified proteins revealed that 3 unique proteins corresponded to serine/arginine rich protein, membrane protein from Streptomyces coelicolor, and one hypothetical protein from Corynebacterium efficiens YS-314, respectively. Culture filtrate proteins from M. massiliense induced the release of pro-inflammatory cytokines from macrophages in a dose-dependent manner but not that from M. abscessus. Taken together, the functional study on the identified proteins uniquely produced from M. massiliense may provide not only the clues for the different pathogensis, but also help develop the diagnostic tools for the differentiation between two mycobacterial species.