Mycobacterium tuberculosis survives within the infected host cells by escaping phagolysosomal fusion and adaptation to hosts' intracellular milieu. Recently, the role of alveolar epithelial cells (AECs) in the survival of intracellular M. tuberculosis has gained importance. It has been reported that M. tuberculosis causes cytotoxicity in AECs, a phenotype attributed to M. tuberculosis virulence that could be due to the differential gene/protein expression of bacilli in these cells. Thus, the present study focused on comparative proteomic analysis of intracellular mycobacteria within macrophages and epithelial cells. Intracellular mycobacteria from infected alveolar epithelial (A549) and macrophage (THP-1) cell lines were harvested, and two-dimensional polyacrylamide gel electrophoresis of mycobacterial proteins was performed. The protein spots of interest were picked and MALDI-TOF/MS was performed to identify proteins. Differential expression of 11 mycobacterial proteins was found in infected AECs, out of which six proteins were confirmed by MS/MS analysis. The identified proteins mainly belonged to functional categories of virulence, detoxification and adaptation, and are known to take part in metabolism and respiration, maintenance of virulence and survival during stress. These proteins were predicted to contain peptide motifs possessing cell-penetration ability. Expression of such proteins by intracellular mycobacteria could be essential for survival and persistence in AECs in its virulent state, thus contributing to disease pathogenesis.