The pathogenic bacterium Francisella tularensis possesses a non-canonical T6SS encoded on the Francisella pathogenicity island (FPI) that is essential for efficient phagosomal escape and access to the cytosolic compartment of infected host cells. Using a global and site-specific phosphoproteomic analysis of F. novicida, we identified a phosphorylated form of IglB, which constitutes with iglA the outer sheath of the T6SS. We show here that substitution of the unique phosphorylated tyrosine (Y139) of IglB to alanine or to the non-phosphorylatable analogue phenylalanine prevents the formation of the sheath-like structures and impairs normal bacterial phagosomal escape. We propose that IglB phosphorylation is involved in the dynamics of assembly-disassembly of the sheath.