Insertion sequences (ISs) exist widely in bacterial genomes, but their roles in the evolution of bacterial antiphage defense remain to be clarified. Here, we report that, under the pressure of phage infection, the IS transposition of into the gene can occur at high frequencies, which endows the mutant mycobacterium with a broad-spectrum antiphage ability. Lsr2 functions as a negative regulator and directly silences expression of a gene island composed of 11 lipid metabolism-related genes. The complete or partial loss of the gene island leads to a significant decrease of bacteriophage adsorption to the mycobacterium, thus defending against phage infection. Strikingly, a phage that has evolved mutations in two tail-filament genes can re-escape from the inactivation-triggered host defense. This study uncovered a new signaling pathway for activating antimycobacteriophage immunity by IS transposition and provided insight into the natural evolution of bacterial antiphage defense.