Alternative splicing is a key event which enables us to generate protein diversity from a limited set of genetic information. Previously we have shown that Mycobacterium tuberculosis (Mtb) which primarily infects the host macrophages and causes genome-wide transcriptional reprogramming in the host and virulent strain of Mtb H37Rv cause massive alteration in the host RNA splicing that affects the host-cell physiology and helps in its better survival in the host. Here using a high-throughput yeast two-hybrid and mass spectrometry approach we show that mycobacterial proteins can access the RNA splicing machinery of the host, thereby playing a critical role in infection-induced perturbation of alternative splicing. We show that infection with H37Rv or drug-resistant clinical strains results in increased expression of truncated splice variants of a large number of protein-coding genes. These splice variants either do not undergo translation or result in truncated/non-functional proteins upon translation. A more detailed mechanistic understanding of this critical regulatory event in macrophages upon Mtb infection could unravel newer ways to target tuberculosis.